Patent Application
20240377319
Illustrative embodiments of the invention generally relate to infra-red spectroscopy and, more particularly, various embodiments of the invention relate to filters that are optically matched to allow measurement of biological samples dispersed in strongly absorbing reference solutions.
Infra-red (IR) absorption spectroscopy is a powerful tool to analyze protein's secondary structures in aqueous solutions. However, the strong absorption of infrared energy in liquid water, particularly over the wavelength range where the most-desirable, Amide-I band is located (1580-1720 cm−1), made such measurements significantly harder. There are a couple of big challenges.
Due to the large IR absorption of water, which in turn significantly impacts the measurement system's signal-to-noise ratio (e.g., SNR), such measurements require a much brighter light source than conventional FTIR. Alternatives include but are not limited to instruments that have broadly tunable Quantum Cascade Laser (QCL) modules.
More importantly, liquid water's absorption coefficient varies greatly over the wavelength measurement region such that the liquid water layer's absorption characteristics are also highly nonlinear over the measurement wavelength range such that differential transmission at each wavelength varies by 150 times (e.g., 150X) across the operating range.
For this reason, each instrument typically incorporates a set of multiple flat response Neutral Density (ND) filters to utilize the full detector dynamic range yet provide enough bit resolution between attenuation levels. This often requires repeating tests multiple times, each with a fixed attenuation ND filter, which in turn results in at least a 3X longer test time and more test sample volume. For any instruments with high coherence light sources like lasers, strong optical interference between optical surfaces poses significant challenges in achieving the optimum data quality. Moreover, such optical interference varies when passing different components (e.g.,. various ND filters) and drift over time at different rates. Therefore, in addition to the data quality, combining test results that were taken with different ND filters (i.e. stitching) poses yet another unique challenge.
In accordance with one embodiment of the invention, a method to measure a liquid analyte with a weak absorbance in a prescribed reference solution with a very high absorbance includes alternatively flowing the liquid analyte and the prescribed reference solution through a fluid chamber in a liquid flow cell. The method further includes emitting an infra-red (IR) light from an IR light source. The method further includes using a matched optical filter, spectroscopically matched to at least a component portion of the prescribed reference solution to filter the IR light from the IR light source to produce filtered light. The matched optical filter is configured to filter at least a portion of the IR light from the IR light source in a substantially inverse manner to the absorbance of the prescribed reference solution. The matched optical filter transmits the filtered light through the fluid chamber to produce a chamber signal. The method further includes using a detector having an optical range to detect the chamber signal.
The chamber signal may include at least one of an analyte signal or a reference signal. The IR light emitted from the IR light source may include a coherent IR light from a tunable optical laser source. The tunable optical laser source may include tunable optical quantum cascade laser (QCL) configured to operate in an IR region.
The prescribed reference solution may include a portion of at least one component, the components comprising a reference fluid, a salt, and a buffer solution. The reference fluid component may include at least one of a water, an alcohol, or an oil. The reference fluid component may be water.
The method may further include providing a shutter. The shutter may be configured to be positioned in an optical beam path of the filtered light such that the filtered light is blocked from reaching the sample cell at predetermined intervals.
The matched optical filter may reduce the reference signal across a spectral region of interest to be maintained within a correctible linear range of the detector. The matched optical filter may be spectroscopically matched to between about 1600 cm−1 to about 1700 cm−1. The matched optical filter may be spectroscopically matched to between about 1580 cm−1 to about 1720 cm−1. The matched optical filter may be spectroscopically matched to between about 400 cm−1 to about 1200 cm−1. The matched optical filter may be spectroscopically matched to between about 3000 cm−1 to about 3700 cm−1. The detector is spectroscopically matched to the spectral region of interest from between about 1600 cm−1 to about 1700 cm−1.
The method may further include controlling the system with a controller that fully automates the liquid analyte and the reference solution handling. The method may further include controlling the system with a controller that fully automates measurement of the analyte signal and the reference signal. The controller may provide automatic and continuous real-time background referencing.
In accordance with another embodiment of the invention, a system to measure a liquid analyte with a weak absorbance in a prescribed reference solution with a very high absorbance includes a tunable optical laser source configured to emit coherent light across a range of wavelengths. The system further includes a liquid flow cell having a sample chamber with a chamber window configured to alternatively receive the liquid analyte and the prescribed reference solution. The system further includes a matched optical filter spectroscopically matched to the prescribed reference solution.
The matched optical filter is configured to substantially flatten the very high absorbance of a prescribed reference solution signal across at least a portion of an optical spectrum. The portion of the coherent light from the tunable optical laser is filtered in a substantially inverse manner to the absorbance of the prescribed reference solution.
The system may further include a detector configured so that the emitted coherent light may be directed toward the matched optical filter so that the emitted coherent light is filtered to pass a filtered light. The filtered light may be directed to pass through the chamber window in the sample chamber where it interacts with either the liquid analyte to produce a liquid analyte spectrum or the prescribed reference solution to produce a prescribed reference solution spectrum. The detector may be further configured to measure the liquid analyte spectrum or the reference solution spectrum.
The system may further include a controller in electrical communication with the system that fully automates the alternatively receiving the liquid analyte and the reference solution. Further, the controller may fully automate measurement of the liquid analyte spectrum and the reference solution spectrum.
The prescribed reference solution may include a liquid water layer having a thickness of between about 22 +/−0.5 um and about 27 +/−0.5 um.
The matched optical filter may be positioned in an optical path between a laser window and a detector window. The matched optical filter may be positioned in the optical path before the chamber window. The matched optical filter may be combined with one or more of a chamber window, a laser window, or a mirror.
The matched optical filter may include a substrate, and a plurality of layers deposited on the substrate that form an interference filter. The substrate may include at least one of a germanium (Ge) window; a calcium fluoride (CaF2) window; a barium fluoride (BaF2) window; a zinc selenide (ZnSe) window; a chalcogenide window; or a silicon (Si) window. The substrate may include a germanium (Ge) window. The substrate may have a wedged shape to reduce interference effects when used in transmission.
The matched optical filter may include an anti-reflection (AR) coating grown on the matched optical filter substrate on the opposite side of the plurality of layers. The AR coating may increase the transmittance of the filtered light by reducing internal reflection of the filtered light from of the inside surface of the matched optical filter substrate. AR coatings may be formed from stacks of transparent thin film structures with alternating layers of contrasting refractive index.
The matched optical filter may be adjusted to compensate for differences in laser gain across the spectral region of interest. The matched optical filter may be configured to provide compensation that is between the reference fluid and the sample fluid. The liquid analyte may include at least one of a protein, a nucleic acid, a lipid-like substance, or a virus.
The tunable optical laser source may include a quantum cascade laser (QCL). The QCL may emit a coherent light in a wavenumber range between 1580 cm−1 to 1720 cm−1. The QCL may operate with a resolution of 1 cm−1. The QCL may operate within an analyte concentration range of between about 0.1 mg/mL to 200 mg/mL. The QCL may operate with a high power output, the high power output being between about 5 mW and 200 mW.
The system may measure an infra-red (IR) absorbance spectrum of the liquid analyte. The system may measure an IR absorbance spectrum of the prescribed reference solution. A ratio of the IR absorbance spectrum of the liquid analyte to the IR absorbance spectrum of the prescribed reference solution is performed to produce a differential absorbance signal.
The alternately receiving of the liquid analyte and the prescribed reference solution in the sample chamber may occur at a rate of between about 0.1 cycles per second and 50 cycles per second without moving the sample cell.
In accordance with yet another embodiment of the invention, a matched optical filter for use with a prescribed reference solution includes a substrate, and a plurality of layers on the substrate that form an optical filter spectroscopically matched to the prescribed reference solution. The matched optical filter may be configured to compensate for a very high absorbance of a reference solution signal across a portion of an optical spectrum, the matched optical filter configured to filter at least a portion of coherent light from a tunable optical laser to a substantially inverse manner to the absorbance of the prescribed reference solution. The matched optical filter may flatten out an optical power of a liquid water layer absorption spectrum measured by a detector after the filtered light passes through a liquid water layer having a thickness of between about 22+/−um and about 27+/−um.
The matched optical filter may flatten out an optical power of a liquid water layer absorption spectrum measured by a detector after the filtered light passes through a liquid water layer having a thickness of between about 10 um and about 100 um.
The substrate may include at least one of a germanium (Ge) window; a calcium fluoride (CaF2) window; a barium fluoride (BaF2) window; a zinc selenide (ZnSe) window; a chalcogenide window; or a silicon (Si) window. The substrate may be germanium (Ge).
The matched optical filter may include an anti-reflection (AR) coating grown on a side of the substrate that is opposite of the side having the plurality of layers. The matched optical filter may include an optical bandpass filter. The matched optical filter may include a tailored bandpass filter. The matched optical filter may include a coated mirror.
Illustrative embodiments of the invention may be implemented as a computer program product having a computer usable medium with computer readable program code thereon. The computer readable code may be read and utilized by a computer system in accordance with conventional processes.
Those skilled in the art should more fully appreciate advantages of various embodiments of the invention from the following “Description of Illustrative Embodiments,” discussed with reference to the drawings summarized immediately below.
In illustrative embodiments, matched optical filters, and methods and systems for using matched optical filters for acquiring infra-red transmission spectra of liquid analytes in very highly absorbing reference solutions are disclosed herein. The matched optical filters have transmission profiles that compensate for the very high absorbance of the reference solution by filtering out at least a portion of coherent light from a tunable infra-red (e.g., IR) optical laser in a substantially inverse manner to the absorbance of the reference solution. The transmission profile of the matched optical filter may be adjusted to compensate for differences in laser gain across a spectral region of interest of the reference solution where the differential transmission of the reference solution at each wavelength may vary by more than 150 times across the operating range. Thus, the matched optical filters make it possible to obtain spectra of the liquid analyte that may have several orders of magnitude lower absorbance than the reference solution. Details of illustrative embodiments are discussed below.
When studying the structure of a protein involved in biological processes, it is advantageous to measure the protein in its native formulation. As regarding proteins in animals, and humans in particular, the native formulations involve water. For example, in biopharmaceuticals, most protein solvents are aqueous with only small amounts of other buffers such as salts, sugars, and fatty acids. Thus, the majority of the solution is water.
Depending on the spectral regions of the protein that are of most interest, it is possible that the water in which the protein is dispersed may interfere with collecting the relevant structural information. Even though the presence of the water may make determining the structure of the protein more difficult, it is important to determine the structure of the protein in the water solvent, because the solvent in which the protein is dispersed affects the secondary structure and stability of the protein. Therefore, it is crucial to establish testing and measurement protocols that limit the optical interference of the background solvent (e.g., water).
For many proteins of interest, a protein secondary structure measurement is typically done in the “Amide I region” of the infrared absorption spectrum which is contained within 1580-1720 cm−1. This region is the most informative for determining secondary structure content such as alpha helix, beta sheet, and beta turn. Structural determination is done by fitting gaussian peaks for each structure under a broader absorption curve. Small changes in the broader absorption curve can cause errors in this fitting process, so any measurement of this area should be done in an accurate and repeatable manner.
In the 1580-1720 cm−1 region of the infra-red (IR) portion of the spectrum, water has a very strong absorption. Liquid water has a maximum absorption of 0.12 AU (e.g., arbitrary units) per micron of pathlength in this spectral region and a minimum absorption of 0.035 AU per micron. The peak absorption of liquid water is at approximately 1645 cm−1. For cell path lengths on the order of 23 um, only one laser photon out of 700 incident on the cell will pass through the fluid. Since AU is a log scale, in transmission at a measurement pathlength of 30 um this would range from 0.00021% T to 8.9% T (where % T is percent Transmission). That is, in order to measure a sample of liquid water with a measurement pathlength of 30 um would require a detector having a dynamic range of >450X. Thus, since buffers and samples are in mostly aqueous solutions, the effect of the presence of water is true for all tests. Furthermore, when trying to measure analytes that are strongly absorbing samples in larger amounts, such as 200 mg/mL, there can be another factor of 30X attenuation. Detectors such as MCT (e.g., mercury cadmium telluride) photovoltaic or photoconductive elements are typically used for such sensitive measurements. The photon flux incident on such a detector must be several times larger than the noise equivalent power of that detector. So, at the peak water absorption the detector must produce a usable voltage signal to be accurately digitized. This may typically be at least 200 mV. At the more transmissive portion of the spectrum this signal would become unusually large (200 mV*450X=90V). Additionally, photodetectors are often non-linear which further compresses the usable dynamic range. Thus, the dynamic range required by a detector that covers this spectral range would be too large and it is therefore important to attenuate the light in the regions of the water absorption that are transmissive and allow light in the less transmissive regions to be allowed to pass through to the detector.
For example, a protein such as HEWL (Hen Egg White Lysozyme) at a concentration of 2 mg/mL will have an absorption of 0.012 AU in a 22 um pathlength cell or 0.0005 AU per micron. The protein absorption is a factor of >200X smaller than the water absorption. When measuring a protein solution such as this, any small residual error in ratioing out the water spectrum can carry over into huge errors in the protein spectrum.
Absorbance is related to the molar absorptivity (e.g., the absorption coefficient) of a material, the concentration of the material (e.g., amount of the material), and the pathlength of a sample chamber. That is, by changing one or more of the concentration of the material or the pathlength of the sample cell, it is possible to change the absorbance of a sample of liquid analyte and reference solution. In addition, the absorptivity of a material is wavelength dependent. Therefore, when designing a matched optical filter it is important to know the absorptivity of the sample (e.g., liquid analyte) and the reference solution, as well as the wavelength range that is to be used to measure the absorption of the liquid in the sample chamber.
For example, when a sample material that is to be measured has a weak absorptivity, it may be necessary to increase one or more of the concentration of the sample, the pathlength of the sample cell, or the intensity of the emitted light.
Therefore, depending on the chemical species of sample material and the reference solution, there may be wide range of pathlengths (e.g., sample chamber sizes) that may be employed. For example, a pathlength may range between about 2 um and about 120 um, or may range between about 20 um and about 30 um, or range between about 22 +/−0.5 um and about 27 +/−0.5 um, or about 22 um and about 27 um, or between about 5 um and about 7 um.
An ideal pathlength depends on the amount of analytes present (a longer pathlength means greater contrast) and the signal-to-noise-ratio (e.g., SNR), since a longer pathlength means fewer photons passing through. However, an excessively long pathlength can cause the water layer to heat up, resulting in a shift in the background, and will consume significantly more sample volume.
In embodiments, a matched optical filter may be designed that optimizes the transmission profile pathlength
The microfluidic cell 15a has two inlet channels, one for the sample fluid and one for the reference fluid, and an outlet channel for waste. The height (e.g., the optical pathlength) of the cell channel is on the order of 23 um. Using gas pressure, sample fluids and reference fluids are alternately pushed through the outlet channel. On/off modulation of the flow fluid from each channel is controlled by a microfluidic valve, one for each path (not shown). Furthermore, in embodiments, pushing the fluids through the outlet channels may be accomplished with other types of pumps, such as, syringe pumps, diaphragm pumps, and the like.
The use of microfluidic techniques have additional advantages. The sample fluids (e.g., liquid analyte) and buffers (e.g., reference fluids) can be changed externally, at will, without hardware changes in the optical path.
Furthermore, with the ability to continuously measure samples, the system also allows for use in an on-line application scenario, where process analytical technology and quality control features can be exploited.
Measurement of the sample fluid transparency (e.g., optical absorption spectrum) is conducted when the sample has fully pushed through the optical interrogation region, flushing out all measurable transparency effects of the reference fluid.
Likewise, the measurement of the reference fluid transparency is made once it has pushed fully through the optical interrogation region, removing all measurable effects of the transparency of the sample fluid.
The modulation of the on/off flow fluid from each channel can be operated at a rate of between about 0.1 cycles per second and 50 cycles per second without moving the sample cell. There are advantages to modulating the fluids as quickly as possible. For example, rapid switching times can reduce drifts in light signal or detector dark background signal, as well as decrease noise terms that have 1/f characteristics.
The use of a tunable laser with the rapid on/off fluid switching times enables the generation of a high optical power at the measurement wavelengths, which can be used to increase sample cell path length. This combination enables an improved signal to noise ratio on spectral measurements relative to more conventional forms of spectroscopy.
The ability to take measurements on a rapid time scale of up to 50 cycles per second provides the opportunity to monitor events as a function of time. For example, studying the kinetics of reactions and processes becomes possible when spectra can be measured at up to 50 cycles per second. It is possible to measure a thermal ramp with a faster temporal resolution. That is, by eliminating the need to switch in and out different ND filters, the use of a matched optical filter in a measurement system allows the measurement of fast events. This is quite important for looking at protein stability, enzyme reactions, and binding events. The ability to monitor kinetically fast chemical reactions and processes is a surprising result of incorporation of the matched optical filter in IR measurements.
The fluid analyzer 10 (e.g., IR fluid analyzer) is used to obtain measurements of optical properties of sample fluids (e.g., biological liquid analytes such as proteins) and reference solutions (e.g., a reference fluid, a salt, and/or a buffer solution) that are alternatively flowed into and out of the sample chamber of the liquid flow cell 14. The liquid flow cell 16 includes the microfluidic chamber and the sample cell (not shown), as described above. In operation, the IR optical source 12 produces an optical beam of desired wavelength and power, and this is directed through the sample chamber of the sample cell toward the optical detector 16. As shown in
Using techniques generally known in the art, the control and processing circuitry uses the values represented by the detector output signals to calculate corresponding optical properties of the fluid sample, such as absorbance, and these optical properties are interpreted as functional indications about the samples (e.g., whether a sample contains a certain compound or a detailed structure). These indications may be used in higher-level assessments according to the testing or research being conducted, again as generally known in the art. In the course of these operations, the control and processing circuitry may transmit raw and/or processed test values to external devices/systems such as the user I/O devices, storage, communications etc.
This description uses the term “beam path” 20 to describe the path of an optical beam from a source to a detector, via the sample cell 15b.
In one embodiment, the optical source 12 may be realized using a mid-IR laser, such as fixed frequency, or tunable QCL lasers. These are tuned to suitable wavelength(s) for measuring analyte(s) of interest, such as the peak of an absorbance feature chosen to minimize background interferences. The optical source 12 (e.g., IR optical source) may be coupled to the sample cell through a matched optical fiber 22.
Generally, measurements are differential in nature. That is, they are based on differences in optical response of samples of interest (e.g., a liquid analyte) and a reference solution (e.g., a prescribed reference solution). A prescribed reference solution may be chosen as a suitable blank, such as pure solvent, a gas, or other suitable reference material or mixture representative of a sample background.
The system may employ one or multiple shutters, as described more below. In one example, a shutter configuration 24 may include a flag that can be moved into and out of the beam path 20. In another example, the shutter 24 may include a chopper wheel on either side of the sample cell which alternatively passes light to the detector 16 or blocks the sample and/or reference beams prior to the sample chamber 15b.
In the measurement of fluids using spectroscopic techniques, minimizing the volume consumed in the measurement may be important, because of cost and/or limited availability of analytes of interest. Techniques that reduce the volume of fluid or quantity of analyte required for the measurement may therefore be advantageous. In many spectroscopic methods, the fluidic cell that contains the fluid under test, and the measurement technique associated with the cell, are important aspects of fluid and sample minimization. Spectroscopic methods typically perform a comparison of sample and background (reference) fluids, with the ratio of the two responses being referred to herein as differential absorbance or diffAU. In many applications, it is also preferred that disposable cells be used, which may be preferable for applications requiring thermal or chemical denaturing of proteins during the testing, for example.
The patents and applications listed in Table 1 provide context on optical measurement techniques of the general type described above. The disclosure of which is incorporated herein, in its entirety, by reference.
In some embodiments, the disclosed spectroscopic methods and systems typically perform a comparison of sample and background (reference) fluids, with the ratio of the two responses being referred to herein as differential absorbance or diffAU. This method requires that spectra from each of the sample (e.g., liquid analyte) and the reference (e.g., a reference solution) are measured on a similar scale to allow a diffAU analysis.
However, as described above, in some IR spectral regions of interest, water may have a >200X stronger absorption than the analyte. Furthermore, depending on the region of interest, the water absorption may also have >450X dynamic range variation of voltage measured by a detector.
For example, in order to measure a secondary structure measurement of a protein, IR absorption spectroscopy is characterized in the “Amide I region” of the infrared absorption spectrum which is contained within 1580-1720 cm−1 spectral region.
A common way to take measurements within the given dynamic range is to repeat tests with commercially available neutral density filters, each of which has a relatively flat response over the wavelength range.
The inventors developed a method of optically leveling the incoming infrared energy passing through a thin liquid water layer using a matched optical bandpass filter. An optical bandpass filter has a transmission profile that allows a specific wavelength band to pass while variably attenuating the light to a specific curve. Such wavelength passband filters can be designed with a center wavelength, passband width, and passband shape to create an inverse of the water absorption shape. The design characteristics (e.g., center wavelength, width, and shape) can be achieved by stacking multiples of thin interference layers on a substrate that is transparent in the desired wavelength range.
Described herein is the development of a matched optical filter that has a transmission profile that is matched to a spectral region for a particular reference solution. The transmission profile of the matched optical filter is configured to substantially flatten the very high absorbance of a prescribed reference solution signal across at least a portion of the optical spectrum of interest (e.g., the region corresponding to the region of interest for the liquid analyte). The portion of the coherent light from the tunable optical laser is filtered in a substantially inverse manner to the absorbance of the prescribed reference solution.
Referring to
In embodiments, an anti-reflection (AR) coating may be deposited on a side of the matched optical filter substrate that is opposite the side with the matched optical filter. An AR coating can increase the transmittance of the filtered light through the filter by reducing internal reflection of the filtered light off of the inside surface back side (e.g. exit side) of the matched optical filter substrate. AR coatings may be formed from stacks of transparent thin film structures with alternating layers of contrasting refractive index.
What the inventors discovered, and what was not apparent previously, is that an optical filter could be matched to the strong absorption of a spectral region of a particular reference solution to substantially flatten the very high absorbance of a prescribed reference solution signal across at least a portion of an optical spectrum. It is surprising that a portion of the coherent light from the tunable optical laser can be filtered in a substantially inverse manner to the absorbance of the prescribed reference solution to allow measurement of a sample and a highly absorbing reference solution in a single measurement without any neutral density filters.
At step 1010, alternatively flow the liquid analyte and the prescribed reference solution through a fluid chamber in a liquid flow cell. A microfluidic controller controls the channels of a fluid delivery system that alternates flow of the liquid analyte and the prescribed reference solution the liquid flow cell. The modulation of the on/off flow fluid from each channel can be operated a rate of between about 0.1 cycles per second and 50 cycles per second without moving the sample cell.
At step 1020, emit an IR light from an IR light source. The emitted IR light may be a coherent IR light from a tunable optical laser source. The tunable optical laser source may be a tunable optical quantum cascade laser (QCL) configured to operate in an IR region. The sample chamber in the microfluidic cell is positioned in a beam path of the IR light that is emitted from the light source and is directed to pass through the sample chamber and proceed to the detector.
At step 1030, use a matched optical filter spectroscopically matched to at least a component portion of the prescribed reference solution to filter the IR light from the IR light source to produce filtered light. The matched optical filter may be configured to filter at least a portion of the IR light from the IR light source in a substantially inverse manner to the absorbance of the prescribed reference solution. The filtered light may be through the fluid chamber to produce a chamber signal.
At step 1040, transmit the filtered light through a sample cell in the fluid chamber to produce a chamber signal. The filtered light may transmit through the sample cell in a transmissive mode or in a reflective mode. In a transmissive configuration, the light flows in a beam path that extends through the sample cell from one side to the other. In a reflective configuration, the light flows in a beam path that is incident at one side of the sample cell and is reflected thereby to a detector arranged on the same side.
At step 1050, use a detector having an optical range to detect the chamber signal. The matched optical filter is spectroscopically matched to at least one of: from between about 1600 cm−1 to about 1700 cm−1; from between about 1580 cm−1 to about 1720 cm−1; from between about 400 cm−1 to about 1200 cm−1; or from between about 3000 cm−1 to about 4000 cm−1. The detector may be spectroscopically matched to the matched optical filter.
In embodiments, an optional step includes providing a shutter. The shutter is configured to be positioned in an optical beam path of the filtered light such that the filtered light is blocked from reaching the sample cell at predetermined intervals.
In further embodiments, another optional step includes controlling the system with a controller that fully automates the liquid analyte and the reference solution handling, and fully automates measurement of the analyte signal and the reference signal. Additionally, the controller may provide automatic and continuous real-time background referencing.
The optional steps may be utilized individually, or in combination with the steps 1010 to 1040, inclusive.
The following examples are intended to further illustrate the disclosure and its preferred embodiments.
Water spectra can change with temperature, and incident light can cause the temperature of the solution to change, therefore changing the spectrum. It's advantageous to do measurements quickly and at the lowest incident power possible.
Measuring lipids in aqueous solution has important applications in biopharma. For example, LNP (lipid nanoparticles) are currently being used as delivery vessels for active ingredients of drug products. The lipids serve as a barrier that protects the drug (typically a protein) until it reaches a target cell. Lipids are made up of fatty acids which have various CH absorption bands in the 2800-3700 cm−1 region. The symmetrical stretching mode of water also overlaps this region which, similar to Amide I, makes precise measurements of solutions/suspensions difficult.
In this embodiment the matched optical filter is designed to be complementary to the OH symmetrical stretching absorption of water in the 3000-3700 cm−1 region.
O—H of water,
N—H of amide,
C—O of carbohydrates
C—H of C═CH— chains of lipids
CH3 of mehtyl groups
CH2 of methylene groups
CH2 and
CH3 of methyl and methylene groups
C═O ester of lipids and fatty acids
C═O of proteins (Amide I)
N—H and
C—N of protiens (Amide II)
CH2 and
CH3 of methyl and methylene groups
CH2 and
CH3 from proteins and
C—O from carboxylic
P═O from phosphodiester of nucleic acids and
C—O—C from polysaccharides
symmetric stretching;
asymmetrical stretching;
symmetric deformation (bend);
asymmetric deformation (bend).
indicates data missing or illegible when filed
The fingerprint region of the infrared spectrum (typically considered 400-1200 cm−1) is very useful for identifying unknown analytes. Academic research has shown this region can be used to identify bacterial strains (1) however the water absorption spectrum overlaps a significant portion of the finger-print region making measuring aqueous solutions challenging.
Similar to above, the same “fingerprint” region could be used to identify cell media components such as metabolites and cellular waste products. For example, lactate has characteristic absorbances in the fingerprint region which the strong water absorption overlaps and interferes with. This problem is pointed out in
It is important to note that the samples measured in
Nucleic acids share absorptions across the infrared spectrum with very informative peaks in the 700-1200 cm−1 region and 1580-1690 cm−1 region. These regions also overlap water absorption bands as shown previously and would benefit from a matched optical filter to reduce the necessary dynamic range.
[14, 16, 11, 9, 12].
of
in ss or ds
of
in ds
of
in ss
in ss
(
wobble)
of G in ds
of G in ss
and
and 1320
conformation (
base pair)
-base paired G (
wobble)
O2of C in ss or ds
)
S-type
N-type
indicates data missing or illegible when filed
The embodiments of the invention described above are intended to be merely exemplary; numerous variations and modifications will be apparent to those skilled in the art. Such variations and modifications are intended to be within the scope of the present invention as defined by any of the appended claims.
