LIMULUS AMOEBOCYTE LYSATE ASSAY AND METHOD OF SAME

Abstract

A pyrogenicity test assay and method of pyrogen testing that allows for rapid and ultrahigh sensitivity testing of parenteral pharmaceuticals or medical devices that contact blood or cerebrospinal fluid by employing a Limulus Amoebocyte Lysate (LAL) assay utilizing a photonic-crystal biosensor. The photonic-crystal biosensor is capable of determining the presence of endotoxins in a test sample by monitoring shifts in the resonant wavelength of an open microcavity affected by the changes in the refractive index of the analyte solutions used.

Claims

1. A Limulus Amoebocyte Lysate (LAL) assay system comprising means for measuring endotoxins in an analyte by monitoring changes in a refractive index of the analyte.

2. The LAL assay system of claim 1, wherein the means for measuring endotoxins comprise a photonic crystal biosensor.

3. The LAL assay system of claim 1, wherein the means for measuring endotoxins comprise a photonic crystal total internal reflection (PC-TIR) biosensor.

4. The LAL assay system of claim 3, further comprising at least one analyte well associated with the PC-TIR biosensor.

5. The LAL assay system of claim 3, further comprising means for providing polarized light to the PC-TIR biosensor.

6. The LAL assay system of claim 4, further comprising a prism for transmitting light to the PC-TIR biosensor and passing the light from the PC-TIR biosensor to a spectrometer for receiving the transmitted light.

7. The LAL assay system of claim 5, further comprising: a light source having a polarizer optically coupled thereto; a beam splitter optically coupled to the polarizer; a prism optically coupled to at least one of a plurality of analyte wells associated with the PC-TIR biosensor; and a spectrometer optically coupled to the prism.

8. The LAL assay system of claim 7 wherein an optical fiber optically couples the light source to the polarizer.

9. The LAL assay system of claim 3, wherein the PC-TIR biosensor further comprises a plurality of microchannels on a surface thereof.

10. The LAL assay system of claim 9, further comprising: a laser light source providing a laser light beam; a polarizer positioned to receive the laser light beam from the laser light source; a spatial filter configured to receive a polarized laser light beam from the polarizer and expand the polarized laser light beam; a plurality of mirrors configured to direct the expanded polarized laser light beam toward the PC-TIR biosensor; a lens configured to focus the expanded polarized laser light beam into a line crossing at least one of the plurality of microchannels on the surface of the PC-TIR biosensor; a collimating lens positioned to receive a reflected laser light beam from the surface of the PC-TIR biosensor; and an imaging device capable of imaging the collimated reflected laser light beam to create an image, the image having a plurality of sections corresponding to a resonant angle of the laser light beam.

11. A Limulus Amoebocyte Lysate (LAL) assay method wherein a refractive index of an analyte is measured.

12. The LAL assay method of claim 11, wherein measuring of the refractive index of the analyte further comprises the step of placing the analyte in an evanescent field of a photonic crystal biosensor.

13. The LAL assay method of claim 11, further comprising the step of providing a photonic crystal biosensor having an open cavity and a total internal reflection.

14. The LAL assay method of claim 13, further comprising the step of polarizing light prior to the step of passing the polarized light through the analyte.

15. The LAL assay method of claim 14, further comprising the step of beam splitting the polarized light prior to the step of passing the light through the analyte.

16. The LAL assay method of claim 11, further comprising the step of measuring statistically significant resonant wavelength shifts within approximately two minutes of starting the LAL assay.

17. The LAL assay method of claim 11, further comprising the step of measuring statistically significant resonant wavelength shifts within approximately five minutes of starting the LAL assay.

18. The LAL assay method of claim 11, further comprising the step of measuring statistically significant resonant wavelength shifts within approximately ten minutes of starting the LAL assay.

19. The LAL assay method of claim 11, further comprising the step of providing an LAL reagent.

20. The LAL assay method of claim 19, wherein the LAL reagent selected comprises Factor C reagent, and wherein the Factor C reagent selected comprises recombinant Factor C.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0015] FIG. 1 is a schematic of the PC-TIR sensor for LAL assays.

[0016] FIG. 2 is a schematic of the inventive PC-TIR sensor based LAL assay system in spectroscopy detection mode.

[0017] FIG. 3 is a schematic of the inventive PC-TIR sensor based LAL assay system in angular detection mode.

[0018] FIG. 4 shows reflectance spectra from a photonic crystal biosensor in an LAL endotoxin assay.

[0019] FIG. 5 is a time dependence graph of resonant dips in reflectance spectra of the PC biosensor in LAL endotoxin assays.

[0020] FIG. 6 is a graph of resonant wavelength shifts of a PC-TIR biosensor at five minutes in LAL assays of endotoxin with different concentrations.

[0021] FIG. 7 is a graph of reaction rate between LAL reagents and endotoxin with different concentrations.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0022] The foregoing and other features and advantages of the invention are apparent from the following detailed description of exemplary embodiments, read in conjunction with the accompanying drawings. The detailed description and drawings are merely illustrative of the invention rather than limiting, the scope of the invention being defined by the appended claims and equivalents thereof

[0023] Embodiments of the invention will now be described with reference to the Figures, wherein like numerals reflect like elements throughout. The terminology used in the description presented herein is not intended to be interpreted in any limited or restrictive way, simply because it is being utilized in conjunction with detailed description of certain specific embodiments of the invention. Furthermore, embodiments of the invention may include several novel features, no single one of which is solely responsible for its desirable attributes or which is essential to practicing the invention described herein. The words proximal and distal are applied herein to denote specific ends of components of the instrument described herein. A proximal end refers to the end of an instrument nearer to an operator of the instrument when the instrument is being used. A distal end refers to the end of a component further from the operator and extending towards the surgical area of a patient and/or the implant.

[0024] The use of the terms a and an and the and similar referents in the context of describing the invention are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. It will be further understood that the terms comprises, comprising, includes, and/or including, when used herein, specify the presence of stated features, integers, steps, operations, elements, and/or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and/or groups thereof.

[0025] Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. The word about, when accompanying a numerical value, is to be construed as indicating a deviation of up to and inclusive of 10% from the stated numerical value. The use of any and all examples, or exemplary language (e.g. or such as) provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any nonclaimed element as essential to the practice of the invention.

[0026] References to one embodiment, an embodiment, example embodiment, various embodiments, etc., may indicate that the embodiment(s) of the invention so described may include a particular feature, structure, or characteristic, but not every embodiment necessarily includes the particular feature, structure, or characteristic. Further, repeated use of the phrase in one embodiment, or in an exemplary embodiment, do not necessarily refer to the same embodiment, although they may.

[0027] As used herein the term method refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, pharmacological, biological, biochemical and medical arts. 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 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.

Biosensor Chip Preparation

[0028] In accordance with the best mode contemplated for the present invention, the PC-TIR biosensor employed in the present invention has an open optical microcavity and is based upon the PC-TIR biosensor described in U.S. Pat. No. 7,639,362, which is herein incorporated by reference in its entirety. The inventive PC-TIR biosensor was designed based on numerical simulations with a transfer matrix approach and fabricated with electron-beam physical vapor deposition. As shown in FIG. 1, the inventive PC-TIR biosensor 300 has a plurality of alternating layers 312 of titanium dioxide and silicon dioxide coated on a glass substrate 310. In accordance with an illustrative embodiment, each TiO.sub.2 layer is about 91 nm in thickness, each SiO.sub.2 layer is about 309 nm in thickness and a glass substrate is BK-7 optical glass. A cavity layer 314 was formed above the plurality of alternating layers and was formed with about 382 nm silica and about 8 nm of silicon. A thin silicon layer gives rise to an appropriate level of absorption such that a sharp dip in the reflectance spectrum is introduced at the resonant wavelength of an open optical microcavity or open cavity formed when the PC structure is used in a TIR configuration (Ye et al. 2013; Ye et al. 2009). When the RI of analyte solutions on top of the PC-TIR biosensor surface changes, the resonant wavelength shifts accordingly. Sample or Analyze wells or microfluidic channels (223 in FIG. 3) can be attached to or formed in the top surface 316 of the PC-TIR sensor 300 for sample handling, which may be formed using a polydimethylsiloxane (PDMS) replica molding process according to one embodiment. PDMS base and curing agents (SYLGARD184, Dow Corning) were mixed at a ratio of about 10:1. The mixture was degassed in a vacuum chamber for about 10 minutes and then cast on a mold and cured at room temperature. To bind the sample wells with the biosensor chip, the surface of a PC-TIR biosensor chip and the PDMS sample well were first processed with a plasma cleaner (Harrick Plasma) for about 60 seconds, which renders the surface hydrophilic. The silanol (SiOH) groups created on the surface form bridging SiOSi bond when the oxidized PDMS surface was placed in contact with the biosensor chip surface, creating an irreversible seal. The PC-TIR biosensor chip together with the sample wells were baked at 200 C. for 1 hour before usage to remove any possible contaminations.

[0029] Biosensor Apparatus

[0030] The inventive LAL assay system 100 in spectroscopy detection mode, as depicted in FIG. 2, was used to sensitively detect the changes in RI of the LAL analyte solutions on the PC-TIR biosensor 222. White light produced by a fiber illuminator 110 (Thorlabs OSL1) as the light source was coupled into a single-mode optical fiber 114 for transmitting light, and S-polarization was selected with a polarizer 116 before the beam was split into two using a beam splitter 120 and mirrors 118. The polarized light then passed with mirrors 122 to the biosensor 124 mounted on an optically transparent prism 126, the biosensor 124 was oriented in a TIR configuration and the reflectance spectra of the two optical paths from the biosensor were detected with a high-resolution spectrometer 138 (Ocean Optics HR 4000). Readings were taken via the Ocean View software package (Ocean Optics) and recorded as full spectrum data files every 30 seconds. Each spectrum consisted of the average of ten 0.1 second scans with boxcar smoothing with a radius of about 2.

[0031] The inventive LAL assay system 200 may be utilized in angular detection mode, as shown in FIG. 3. For this embodiment, a laser 210 is the laser light source, such as a Helium-Neon laser, and is used as a probe light to monitor changes in the resonant condition of the PC-TIR biosensor 222 due to reaction of LAL reagents with endotoxins. The laser light source transmits a laser light beam, which first goes through a polarizer 212 to select s-polarization for the sensor mounted on a prism. The polarized laser light beam then passes through a spatial filter composed of a pinhole 216 and a pair of lenses 214, 217 to obtain a high quality spatial mode and expand the laser light beam 219, preferably to about 10 mm. Two mirrors 218 were used to direct the laser light beam 219 towards the PC-TIR biosensor 222 coupled to an optical prism, preferably with index matching oil. A cylindrical or collimating lens 220 is used to focus the laser light beam 219 into a line crossing a plurality of microchannels 223 on the PC-TIR biosensor 222 surface. The reflected laser light beam 225 is collimated with another cylindrical lens 226 and an imaging device 228 images the collimated reflected laser light beam, which may be a CMOS camera in one embodiment. A plurality of short sections of dark lines appear on the image 230, which correspond to the number of the plurality of microchannels 223 and the resonant angle of the probe beam 219 for the microchannels 223 on the PC-TIR biosensor 222. The positions of the dark lines sensitively shift due to the change of refractive index of the test samples in the microchannels when LAL reagents react with endotoxins in the samples.

Limulus Amoebocyte Lysate (LAL) and Endotoxin Preparation

[0032] In one embodiment, an LAL reagent KTA2 (Charles River, lot D2161L, 0.005 EU/mL sensitivity) was used. KTA2 is a kinetic turbidimetric reagent. The directions for proper rehydration and preparation included in the LAL reagent package were followed. The control standard endotoxin (Charles River, CSE, Catalog # E120) was employed. Solutions of 0.05 EU/mL, 0.0025 EU/mL, 0.00025 EU/mL, and 0.000025 EU/mL were made by dissolving the appropriate weight of endotoxin in LAL reagent water (LRW) (Charles River), according to the CSE instructions.

Measurement Procedure

[0033] After the appropriate dilutions of CSE were made and LAL reagents were rehydrated, the baked biosensor was placed on an equilateral prism with index matching fluid and about 150 l of LRW was first added into both sample or analyte wells on the biosensor. Alignment of the optical beam path reflected from the biosensor was conducted to couple the probe beam into a high-resolution spectrometer, so that resonant dips appeared at the correct wavelength in the reflectance spectrum of the biosensor. The LRW was then replaced with about 150 l of rehydrated Lysate in the sample wells of the biosensor. Next, 150 l LRW was added into one well and the timer was started to record data in about 30 second increments, while about 150 l of the endotoxin sample was added into the other well. Detection of the spectrum of the reflected beams from the two wells was switched every thirty seconds, so that the spectrometer measured the spectra of both wells every minute. Each run was monitored for about 20 minutes. After the assay, the sample wells and the biosensor chip were cleaned first with acetone, and then with deionized water, and dried with compressed air to assure no fluid solution remained on the biosensor surface. In order to reuse the biosensor, the cleaned biosensor chip together with the sample wells were baked at about 200 C. on a hot plate or in an oven for at least one hour in an aluminum enclosure, to ensure even heat transfer, and to restrict room air contamination.

[0034] Triplicates of all solutions were run to make statistical analysis possible. Because each data point consisted of the average of about 10 spectral samples, the standard error was selected as the appropriate error bar in figures showing run results. Standard deviation and the coefficient of variation were also calculated using Excel. Adjusted R-square values were used to judge the goodness of fit for the time-dependent data of the resonant wavelength shifts of a PC-TIR biosensor. Biosensor drift was corrected for by subtracting the H.sub.2O only data from the other data sets.

[0035] The term statistically significant may relate to the coefficient of determination, denoted R.sup.2, which is the proportion of the variance in the dependent variable that is predictable from the independent variable(s). Thus, in this case, statistically significant resonant wavelength shifts the R.sup.2, value around 0.90 and 0.99 would be statistically significant.

EXAMPLES

[0036] 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. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

[0037] 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.

[0038] Results

[0039] In previous studies PC-TIR sensors were used for molecular binding assays ranging from well-studied coupling agents to small molecule binding and nucleic acid and cardiac biomarker detection (Guo et al. 2010; Guo et al. 2008; Zhang et al. 2011; Zhang et al. 2014; Zhang et al. 2013; Zhang et al. 2016). In contrast, for the first time, the present invention utilizes the PC-TIR biosensor to monitor the changes in RI of LAL analyte solutions, which sensitively reflects the amount of endotoxin in the test samples. FIG. 4 shows several representative reflectance spectra of a PC-TIR biosensor at different time points when a LAL analyte solution was added on the biosensor surface. The sharp resonant dip shifts with time while the LAL responds to the endotoxin.

[0040] The wavelength shift of the resonant dip in the reflectance spectra of the PC-TIR biosensors was monitored with a high-resolution spectrometer for every minute over a 20-minute period for each sample. FIG. 4 illustrates the wavelength shift over time. The reflectance spectra were loaded into the OriginPro data analysis program by OriginLab to apply a Lorentzian curve fit and determine the center wavelength of the resonant dip. By fitting the curve rather than simply picking the lowest point of the spectrum, one can obtain a more accurate value of the resonant wavelength. The time dependence of the center resonant wavelength obtained from fitting the spectral curve is shown in FIG. 5. The results shown are for the test samples with different concentrations of endotoxin as well as the control samples. The error bars represent the standard errors of different runs. The data indicate that even the most diluted endotoxin used (about 0.000025 EU/mL) resulted in a clearly measurable difference from the negative control (LAL with H.sub.2O). Based on the average pyrogenicity of endotoxin this equates approximately to about 0.0000025 ng/mL of endotoxin in the solution, which is by far the greatest sensitivity of any previously published LAL testing strategies. Given that the LAL reagent employed has a sensitivity of about 0.005 EU/mL, the system and method of the present invention demonstrates at least a 200-fold increase in detection sensitivity. This significant improvement in detection sensitivity can be attributed to the PC-TIR's capability for ultrasensitive measurements of the RI changes in the analyte solutions caused by LAL reactions to the minute amount of endotoxin. The sharp resonance bandwidth (2 nm) of the PC-TIR biosensor shown in FIG. 4 is much narrower than a typical surface plasmon resonance (SPR) biosensor (40 nm) (Hoa et al. 2007; Homola 2003, 2008), thus allowing precise measurements of resonant wavelength shifts for enhanced detection sensitivity.

[0041] In addition to the sensitivity, the time required for LAL assays is also an important consideration in industry applications for higher throughput and cost saving. To illustrate the fast response of the PC-TIR biosensor to LAL assays, the resonant wavelength shift of the biosensor has been plotted as a function of the endotoxin concentration at a selected time point-5 minutes after endotoxins were added to LAL reagents. FIG. 6 shows that the shift in the resonant wavelength of the biosensor increases with increasing the concentration of the endotoxin and within this short five minute period, all the samples with different endotoxin concentrations can already be well distinguished. This result clearly demonstrates the fast response time, another unique feature of the PC-TIR biosensor based LAL assays in addition to its ultrahigh sensitivity.

[0042] It can be seen from FIG. 5 that different amounts of resonant wavelength shifts appear even as early as two minutes, although the 5-minute time point was selected for a conservative claim. In contrast, the conventional approaches, including gel clot, turbidimetric, and chromogenic assays, take much longer to generate assay results, especially for samples with low endotoxin concentrations. Gel clot assays typical take one hour, while turbidimetric and chromogenic assays also need about 15 minutes to complete one run. The fast response of the PC-TIR, even without the need for equilibrating to about 37 C., can significantly shorten the assay time, which is desired in many industry applications.

[0043] To obtain the reaction rate of the LAL assays detected with the PC-TIR biosensor, a nonlinear least squares regression was used to analyze the time dependent resonant wavelength shifts of the biosensor, which can be fitted to the following first order exponential function (1)

=A(1e.sup.Bt) (1)

where A and B are fitting parameters. The fitting curves are plotted in FIG. 5 while the fitting parameters are listed in Table 1 for test samples with different endotoxin concentrations.

[0044] Table 1. Fitting results of the time dependent resonant wavelength shifts for test samples with different concentrations of endotoxin.

TABLE-US-00001 Fitting Parameters + Sample Value Adjusted R.sup.2 LAL + Endotoxin A = 4.775 0.163 0.994 (0.05 EU/mL) B = 0.081 0.005 LAL + Endotoxin A = 2.355 0.088 0.984 (0.0025 EU/mL) B = 0.106 0.008 LAL + Endotoxin A = 1.793 0.108 0.964 (0.00025 EU/mL) B = 0.074 0.008 LAL + Endotoxin A = 1.537 0.414 0.958 (0.000025 EU/mL) B = 0.040 0.015

[0045] The adjusted R square values obtained from the fitting are close to one, which indicates the goodness of fit. By taking the derivative of Eq. (1), the reaction rate was obtained according to formula (2):

Reaction Rate=ABe.sup.Bt (2)

which is plotted in FIG. 7. It can be seen that the reaction rate is generally higher for higher concentrations of endotoxin samples. Thus, the reaction rate at certain time points, for example at t=0, may also be utilized as a good parameter in addition to the resonant wavelength shifts to indicate endotoxin concentrations. Moreover, there is a general trend that the reaction rate for each sample decreases with assay time. This is reasonable as the reaction between LAL reagents and endotoxins reduces the amount of LAL reagents or free endotoxins and slows down the reaction.

[0046] It has also been found that in addition to using LAL reagents, extracts from LAL reagents that include purified Factor C and/or recombinant Factor C (rFC) may be employed with the present invention. LAL testing may not include protease amplifying steps, thereby necessitating higher sensitivity biosensor be employed. The increased sensitivity of the PC-TIR biosensor in the present system and method, relative to that of conventional LAL assay systems and methods, addresses this need for higher sensitivity.

[0047] The inventive system and method demonstrate that an LAL assay that measures the refractive index of a test solution using a photonic crystal biosensor offers significantly enhanced sensitivity together with substantially faster discrimination of positive and negative results. The PC-TIR biosensor chips can be reused as they are robust through a number of heat cycles. This improvement can be attributed to the new detection principle based on a PC-TIR biosensor for sensitive monitoring of RI changes in the analyte solutions, which is fundamentally different from other conventional methods of LAL assays. In addition, the PC-TIR biosensor has a simple 1-dimensional (1D) PC structure, which is basically a multi-layered dielectric coating and can be easily fabricated with well-established vacuum deposition techniques. In contrast to 2D or 3D PC structures, the simplicity of the PC-TIR biosensor structure allows for low-cost reproduction, robust performance, and ultrahigh sensitivity assays, which are important factors for potential commercialization of this unique technique for sensitive and rapid LAL assays.

[0048] Furthermore, there is promise in testing samples that possibly have confounding variables via extreme dilution due to the fact the LAL assays on a PC-TIR chip have a very high sensitivity. Future research is needed in this area, and a more complete list of confounding molecules can be tested with the PC-TIR chip with the endotoxin standard, blood plasma, and other analytes of interest. A method for binding the LAL proteins to the biosensor surface in conjunction with a microfluidic system may further lead to lower volumes of LAL needed to test solutions for pyrogenicity.

[0049] All publications and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

[0050] While the invention has been described in connection with various embodiments, it will be understood that the invention is capable of further modifications. This application is intended to cover any variations, uses or adaptations of the invention following, in general, the principles of the invention, and including such departures from the present disclosure as, within the known and customary practice within the art to which the invention pertains.