Filtration for bacteria antibiotic susceptibility analysis and internal standard

Abstract

The present disclosure relates to filters configured for a spectroscopic antibiotic susceptibility analysis and a method for filtering a sample for a spectroscopic antibiotic susceptibility analysis. In one example, a method comprises providing a solution comprising bacteria incubated in a media to a well, filtering the solution via adsorption to trap at least a portion of the bacteria in a filter separate from the media. providing a solution of SERS active nanoparticles to the well, and spectroscopically analyzing the filter to determine the presence and / or absence of at least one of a purine and a pyrimidine. Methods of providing an internal standard in a spectroscopic antibiotic susceptibility test are also provided.

Description

FILTRATION FOR BACTERIA ANTIBIOTIC SUSCEPTIBILITY ANALYSIS ANDINTERNAL STANDARDCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of United States provisional application no. 63 / 675,476, filed 25 July 2024 entitled “Filtration for bacteria antibiotic susceptibility analysis” and of United States provisional application no. 63 / 686,077 filed 22 August 2024 and entitled “Filtration for bacteria antibiotic susceptibility analysis and internal standard”, each of which is hereby incorporated by reference as though fully set forth herein.BACKGROUNDField

[0002] The instant disclosure relates to fdtration for antibiotic susceptibility analysis and internal standards useful therein.Background

[0003] Sepsis, the body’s inflammatory response to a bacterial blood infection, is a serious health problem that leads to tissue damage, organ failure and death without timely and targeted antibiotic treatments. In 2017 ~20% of all deaths worldwide were sepsis related. 1.7 M develop sepsis in the US annually and -16% die as a result. Growth based procedures can take 2 - 3 days for AST results and initial broad-spectrum antibiotics lead to poor outcomes and promotes the growing threat from drug resistant strains. Each hour without best treatments leads to increased mortality. Aside from greatly improved health outcomes and enhanced antimicrobial stewardship, significant cost savings (shorter hospital stays, fewer longer term health complications, less tech time, etc.) can only be realized by use of an automated platform that shortens AST for bloodstream infections.

[0004] In a 2023 study by *Brandon M Carius, et al., they found that patients included US military (34%), (non-NATO) military (33%) and humanitarian (30%) groups diagnosed with sepsis had a significantly lower survival rate than non-septic patients (78.1% vs. 95.7%, p < 0.001). Their conclusion was sepsis occurred infrequently in the DoDTR (Department of Defense Trauma Registry) from 2007 to 2020 when evacuation from battlefield is not delayed, but despite increased intervention frequency, developing sepsis demonstrates a significant drop in survival rates.BRIEF SUMMARY

[0005] In one embodiment, a method for filtering a sample for a spectroscopic antibiotic susceptibility analysis is provided. The method comprises providing a solution comprising bacteria incubated in a media to a well, filtering the solution via adsorption to trap at least a portion of the bacteria in a filter separate from the media, providing a solution of SERS active nanoparticles to the well, and spectroscopically analyzing the filter to determine the presence and / or absence of at least one of a purine and a pyrimidine.

[0006] The operation of filtering the solution may comprise at least one wash of the bacteria solution through the filter, and in some instances the operation of washing the solution may comprise washing the solution with distilled water to initiate a starvation response in the bacteria.

[0007] In one embodiment, the operation of filtering the solution comprises passively filtering the solution through the filter.

[0008] The adsorptive filter and / or the media in some embodiments may comprises a purine free filter and / or lack a spectroscopic feature in common with at least one of a purine and a pyrimidine of interest.

[0009] In one embodiment, the filter comprises a purine-free filter.

[0010] In one embodiment, the media comprises a purine-free media, such as casein hydrolysate.

[0011] In one embodiment, the solution comprises different concentrations of at least one antibiotic.

[0012] In one embodiment, the filter and / or the media lacks a spectroscopic signal peak corresponding to at least one spectroscopic signal peak of the at least one of the purine and the pyrimidine.

[0013] In one embodiment, a mechanical barrier at least partially surrounds the filter to reduce or prevent lateral flow of the bacterial solution and / or a wash fluid.

[0014] In one embodiment, the method can identify the bacteria based upon an intensity of the spectroscopic analysis of the at least one of the purine and the pyrimidine.

[0015] In one embodiment, a spectroscopic assay device is provided comprising a plurality of wells; a plurality of adsorptive filters each disposed adjacent to one of the plurality of wells; and a plurality of mechanical barriers each surrounding one of the plurality of filters and configured to prevent lateral flow of a bacterial solution and / or a wash fluid outside of each of the plurality of filters.

[0016] In one embodiment, each of the plurality of adsorptive the filters lacks a spectroscopic feature in common with at least one spectroscopic feature of the at least one of the purine and the pyrimidine. In one embodiment, the filter is a purine-free filter and / or the filter lacks a spectroscopic signal peak corresponding to at least one spectroscopic signal peak of the at least one of the purine and the pyrimidine.

[0017] In another embodiment, a spectroscopic assay device comprises a well plate defining a plurality of wells each comprising a first inlet opening and a second outlet opening; at least one adsorptive filter comprising a first inlet side and a second outlet side, the first inlet side disposed across each of the plurality of second outlet openings of the plurality of wells; and a bottom plate disposed opposing the second outlet opening of each of the plurality of wells and at least partially enclosing the adsorptive filter between the well plate and the bottom plate.

[0018] In one embodiment, at least one of the bottom plate and the well plate comprise at least one mechanical barrier, such as a protrusion, projection, or knife-edge, pinching a portion of the adsorptive filter between the bottom plate and the well plate. The at least one mechanical barrier can reduce or eliminate flow within the adsorptive filter past the at least one mechanical barrier. The at least one mechanical barrier can extend around a perimeter of the well to reduce flow within the adsorptive filter beyond the at least one mechanical barrier.

[0019] In one embodiment, the filter is a purine-free filter and / or the filter lacks a spectroscopic signal peak corresponding to at least one spectroscopic signal peak of the at least one of the purine and the pyrimidine.

[0020] In another embodiment, any of the methods or spectroscopic assay devices may use an intensity of a reference peak, a wavenumber of a spectroscopic analysis, or a peak of a spectroscopic analysis of a component of a spectroscopic assay device, such as a filter, well, well plate, structure, or well is used as an internal standard

[0021] The foregoing and other aspects, features, details, utilities, and advantages of the present invention will be apparent from reading the following description and claims, and from reviewing the accompanying drawings.BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 illustrates an example starvation pathway of bacteria.

[0023] Figure 2 illustrates the molecular structure of a purine species and a strong chemical adsorption to the surface of a SERS-active nanoparticle through their multiple nitrogen bonds.

[0024] Figure 3 illustrates steps to introduce bacteria to cultures with differing amounts of antibiotics and permit incubation.

[0025] Figure 4 illustrates an example filter holder configuration for a syringe.

[0026] Figure 5 illustrates one example embodiment of a filtration approach.

[0027] Figure 6 illustrates Raman spectra of three example filter materials and the spurious signal that stems from the MHB used as a growth medium.

[0028] Figure 7 illustrates an example in which a filter appears to have effectively removed the effect of the MHB growth medium on the spectroscopic analysis of the filter and sample.

[0029] Figure 8 illustrates another embodiment of a well assay cartridge that includes a mechanical barrier to seal the filter and an absorptive material under the well.

[0030] Figure 9 illustrates SERS spectra of a casein hydrolysate and Mueller Hinton Broth (MHB).

[0031] Figure 10 illustrates an example of a basic workflow of an AST process.

[0032] Figure 11 illustrates an example filtering step to remove broth from samples.

[0033] Figure 12 illustrates example steps of washing the pathogens with distilled water to initiate a starvation response and the addition of SERS active nanoparticles to the well.

[0034] Figure 13 illustrates raw data collected from the process shown in Figure 10.

[0035] Figure 14 illustrates data from Figure 4 processed with a nylon reference peak.

[0036] Figure 15 illustrates a plurality of potential intensity reference peaks that could be used for calibration.

[0037] Figure 16 illustrates examples and a SERS spectrum of adenine.

[0038] Figure 17 illustrates spectra of adenine produced by starved E. Coli KI 2.

[0039] Figure 18 illustrates illustrates a spectrometer 100 configured to take a spectroscopic reading of a a sample adsorbed to a filter of a single well 110 (such as shown in Figure 5) and / or a plurality of spectroscopic readings of samples adsorbed to a plurality of filters of a multi-well plate (such as shown in Figure 8).DETAILED DESCRIPTION

[0040] The following description of the invention is provided as an enabling teaching of the invention in its best, currently known embodiment. To this end, those skilled in the relevant art will recognize and appreciate that many changes can be made to the various aspects of the invention described herein, while still obtaining the beneficial results of the present invention. It will also be apparent that some of the desired benefits of the present invention can be obtained by selecting some of the features of the present invention without utilizing other features. Accordingly, those who work in the art will recognize that many modifications and adaptations to the present invention are possible and can even be desirable in certain circumstances and are a part of the present invention. Thus, the following description is provided as illustrative of the principles of the present invention and not in limitation thereof.

[0041] As used throughout, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a” component can include two or more such components unless the context indicates otherwise. Also, the words “proximal” and “distal” are used to describe items or portions of items that are situated closer to and away from, respectively, a user or operator such as a surgeon. Thus, for example, the tip or free end of a device may be referred to as the distal end, whereas the generally opposing end or handle may be referred to as the proximal end.

[0042] All directional references (e.g., upper, lower, upward, downward, left, right, leftward, rightward, top, bottom, above, below, vertical, horizontal, clockwise, and counterclockwise) are only used for identification purposes to aid the reader’s understanding of the present invention, and do not create limitations, particularly as to the position, orientation, or use of the invention. Joinderreferences (e.g., attached, coupled, connected, and the like) are to be construed broadly and may include intermediate members between a connection of elements and relative movement between elements. As such, joinder references do not necessarily infer that two elements are directly connected and in fixed relation to each other.

[0043] 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.

[0044] As used herein, the terms “optional” or “optionally” mean that the subsequently described event or circumstance may or may not occur, and that the description includes instances where said event or circumstance occurs and instances where it does not.

[0045] The term “substantially” as used herein may be applied to modify any quantitative representation which could permissibly vary without resulting in a change in the basic function to which it is related.

[0046] Bacteria undergo a rapid starvation response when placed in a nutrient free medium, for example, distilled water. Figure 1 shows an example starvation pathway of bacteria from Carius BM, Bebarta GE, April MD, Fisher AD, Rizzo J, Ketter P, Wenke JC, Salinas J, Bebarta VS, Schauer SG. A Retrospective Analysis of Combat Injury Patterns and Prehospital Interventions Associated with the Development of Sepsis. Prehosp Emerg Care. 2023;27(l): 18-23. doi:10.1080 / 10903127.2021.2001612. Epub 2021 Dec 22, PMID: 34731068. In one example, rRNA and tRNA are no longer needed for cell growth (it is a starvation response) and they are downgraded to their purine bases and 5 ribose phosphate (5RP) for amino acid production.

[0047] PCT / US24 / 55801 entitled “System and Method for SERS-based AntimicrobialSusceptibility Test” and filed on November 13, 2024 by Ziegler and Carron describe that the purine can be detected by a technique known as Surface Enhanced Raman Scattering (SERS), which is incorporated by reference as if fully set forth herein. SERS uses SERS-active nanoparticles (e.g.,gold, silver, copper, or other nanoparticles) to enhance a Raman signal by as much as 107when the analyte of interest is adhered to the nanoparticle surface.

[0048] Figure 2 illustrates the molecular structure of a purine species and a strong chemical adsorption to the surface of a SERS-active nanoparticle (gold nanoparticles in this example) through their multiple nitrogen bonds. The spectrum (right-hand side) of adenine shows a very strong signal around 733 cm'1wavenumbers. The intensity of the purine signal decreases dramatically in the presence of an antibiotic which the bacteria are susceptible to, likewise in the presence of an antibiotic that the bacteria has become resistant to the signals are unaffected. This is the spectrochemical basis from an AST (Antibiotic Susceptibility Test).

[0049] In one example, a method of performing an Antibiotic Susceptibility Test is shown in Figure 3. Figure 3 shows steps to introduce the bacteria to cultures with differing amounts of antibiotics and permit a 30-minute incubation. This step is followed by multiple centrifugations to remove the growth medium. This has two roles:• To initiate a starvation response by removing all potential food; and• To remove the broth which can interfere with the spectroscopic analysis.

[0050] In one embodiment, a cartridge style medical assay (e.g., a disposable cartridge style medical assay) is provided to simplify or eliminate the requirement of the centrifugation steps.

[0051] As an alternative to centrifuges, bacteria can be filtered. Filter pads of the appropriate porosity (typically 0.45 to 0.22 micron pores) are made of many types of materials and are usually contained in a holder to fit onto a syringe to push the liquid through the filter, likewise, they can be placed in an encapsulating holder and a vacuum can be used to pull the solution from around the bacteria. Figure 4 illustrates an example filter holder configuration for a syringe.

[0052] In one embodiment, a passive filter system is provided in which the sample is filtered without the requirement of an active component such as a syringe push or vacuum draw. An absorptive pad, for example, may be used to draw the solution through the filter by capillary forces.

[0053] Figure 5 shows one example embodiment of a filtration approach using a nylon washer. The washer, in this example, is covered with a black coating to avoid any laser damage, the bottom has an attached porous filter (e.g., attached by waterproof glue). To filter the bacteria the washer assembly is placed on an absorptive pad which is placed on a hard plastic backing.

[0054] In this embodiment, a nylon washer is attached to a porous filter (e.g., glued, clamped or otherwise secured to the porous filter). The washer assembly, in one embodiment, is attached to an absorptive pad (e.g., clamped, glued or otherwise secured to the porous filter). The absorptive pad, for example, can be disposed on the opposite side of the filter from the well defined by the washer.

[0055] The assembly is used to filter off the broth (e.g., Mueller Hinton Broth) and to initiate a starvation response in the bacteria within the well. A filter with 0.45 micron pores, for example, can block the SERS-active nanoparticles for spectroscopic analysis of the sample on the filter.

[0056] In one example, a 5 pL solution of Au colloidal particles was placed on a 0.45 micron pore size filter, and the nanoparticles were trapped on top of or within the filter. This was tested for three properties:• Does the filter material interfere with the Raman signal of the purines and / or pyrimidines?• Does the filter material effectively pass the broth (Mueller Hinton Broth (MHB) in this example)?• Does the filter material hold and permit the starvation response to be detected by spectroscopic analysis of the SERS-active nanoparticles (e.g., gold (Au) or silver (Ag) nanoparticles)?

[0057] The embodiment shown in Figure 5 provides a passive filter system. The filter comprises a structure (e g., the washer or another structure defining a well) comprising a side wall. The side wall defines a well chamber, a first inlet opening, and a second outlet opening. An adsorptive filter (e.g., a nylon submicron filter) comprises a first side and an opposing second side. The first side is disposed adjacent to the second outlet opening of the well and forms a bottom outlet surface of the well. Anan absorptive layer is disposed adjacent to the opposing second side of the adsorptive filter. The absorptive layer is configured to draw a liquid sample from the well through the adsorptive filter, such as via capillary action. The absorptive layer can be configured to draw the liquid sample to adsorb at least one portion of the liquid sample to the adsorptive filter, such as one or more purine, pyrimidine and / or bacteria.

[0058] In one embodiment, a bottom plate is disposed opposing the second outlet opening and at least partially enclosing the adsorptive filter and the absorptive layer between the side wall andthe bottom plate. At least one of the bottom plate and the side wall may comprise at least one mechanical barrier, such as a protrusion, projection, knife edge, or the like, pinching a portion of the absorptive layer between the bottom plate and the side wall. The mechanical barrier is configured to reduce or eliminate flow within the absorptive layer past the at least one mechanical barrier. In one embodiment, for example, the at least one mechanical barrier extends around a perimeter of the well to reduce or eliminate flow within the absorptive layer beyond the mechanical barrier.

[0059] The adsorptive filter and / or the absorptive layer in some embodiments may comprises a purine free filter and / or lack a spectroscopic feature in common with at least one of a purine and a pyrimidine of interest.

[0060] Figure 6 illustrates Raman spectra of three example filter materials and the spurious signal that stems from the MHB used as a growth medium. If the MHB is not effectively removed by washing the MHB through the filters, the MHB residue will exhibit a large peak that corresponds to a spectrographic analysis of the purines. In this example, Figure 6 shows that of the three materials tested, PTFE has a Raman feature in the same region as the purines of interest that could interfere with the detection of the purines. These are not the only three filter materials that are available and are shown as examples.

[0061] The bottom spectrum, labelled Adenine (SERS) is from pure MHB.

[0062] Figure 7 illustrates an example in which a nylon filter with 0.45 micron porosity appears to have effectively removed the effect of the MHB growth medium on the spectroscopic analysis of the filter and sample. The top spectrum labeled Nylon Filter shows the absence of a peak due to adenine. The middle spectrum labeled Nylon Filter + MHB +AuNP illustrates that the SERS spectrum produced by the AUNP exhibits a signal due to the adenine present int the MHB. The third spectrum labeled Nylon Filter + MHB + Washed + AuNP shows that the wash of three volumes of the cell created within the nylon washer removed the MHB. Finally, the bottom spectrum labelled Nylon Filter + E Coli + washed + AuNP illustrates that the starvation response of the purine (adenine) was elicited and once again the purine spectrum of adenine was observed.

[0063] This experiment shows that a nonporous material used to make a well for filtration can be used to purify microbes and to elicit a detectable starvation response through detection of SERS from SERS-active nanoparticles trapped in the well.

[0064] The well material, in this case nylon, can be other nonporous materials. In one embodiment, the materials are not reactive with the solutions, and they wash easily. A hydrophobic material like Teflon or PEEK can be used in one embodiment. While three example filter materials are shown, these are available in a range of polymers and others can be considered. The basic properties are hydrophilicity to permit the aqueous solutions to easily pass through and the lack of Raman features that overlap with the analytical signal of the purines or other target analytes.

[0065] Figure 8 illustrates another embodiment of a well assay cartridge that includes a mechanical barrier, such as but not limited to a protrusion, projection, a knife edge, or the like, to seal the filter and an absorptive material under the well. The mechanical barrier (e.g., knife edge) prevents or reduces lateral flow of solution along (around) the filter material. In this embodiment, the cartridge includes Delrin as the plastic for the wells, an aluminum bottom plate with knife edges to seal the filters around each of the wells, and a bottom aluminum plate with threads to permit the assembly to be clamped together.

[0066] The embodiment shown in Figure 8 comprises a spectroscopic assay device comprising a well plate defining a plurality of wells each comprising a first inlet opening and a second outlet opening. At least one adsorptive filter comprises a first inlet side and a second outlet side. The first inlet side is disposed across each of the plurality of second outlet openings of the plurality of wells. At least one absorptive layer is disposed adjacent the second outlet side of the at least one adsorptive filter, and the at least one absorptive layer is configured to draw a liquid sample from the well through the adsorptive filter. In one embodiment, for example, the absorptive layer is configured to draw the liquid sample to adsorb at least one portion (e.g., at least one of a purine, pyrimidine and / or bacteria of interest) of the liquid sample to the adsorptive filter.

[0067] A bottom plate can be disposed opposing the second outlet opening of each of the plurality of wells and at least partially encloses the adsorptive filter and the absorptive layer between the well plate and the bottom plate. At least one of the bottom plate and the side wall comprise at least one mechanical barrier (e.g., a protrusion, projection, knife-edge, or the like) applying pressure (e.g., pinching) to a portion of the absorptive layer between the bottom plate and the side wall and can be configured to reduce and / or eliminate flow within the absorptive layer past the at least one mechanical barrier.

[0068] The adsorptive filter and / or the absorptive layer in some embodiments may comprises a purine free filter and / or lack a spectroscopic feature in common with at least one of a purine and a pyrimidine of interest.

[0069] Broth Materials

[0070] As stated above, the filter can be used to effectively remove the broth to start the starvation response and to eliminate spectral interference from the broth (e.g., MHB). It is also possible to find off-the-shelf broths or synthetically create fast growing broths that do not inherently contain purines (e.g., use a purine-free broth). Just as one example, Figure 9 illustrates SERS spectra of a casein hydrolysate and Mueller Hinton Broth (MHB). Thes strong peak of MHB that corresponds with a spectral feature of purines, such as adenine, is shown. That peak is absent in the casein hydrolysate broth. The casein hydrolysate broth is rich in amino acids and small peptides for bacterial growth. But analysis shows that it does not contain spectral features that would interfere with the detection of purines, unlike MHB.Methods of Measuring Optical Signals

[0071] In one embodiment, a medical device is configured to measure an Antibiotic Susceptibility Test (AST) to determine if an infective pathogen is susceptible or resistant to specific antibiotics and comprises a calibration method to ensure that the device is providing accurate information. As described above, one method to measure an AST is to use Raman spectroscopy to look at the purine production when a pathogen is placed in a nutrient free environment. In this case the spectrum of the purines can be enhanced with Surface Enhanced Raman Scattering (SERS) active nanoparticles to produce a large distinct signal. When a pathogen is susceptible to an antibiotic, they exhibit a universal response of producing purines when starved and not producing purines when above the level where they are susceptible to an antibiotic. Pyrimidines are also produced and could be used, but they tend to have significantly lower Raman cross-sections.

[0072] To determine this response, one embodiment of a device and method measures the change in a Raman signal due to the adsorption of purines and / or pyrimidines onto SERS active nanoparticles. Optical spectroscopies can be divided into two groups: ratiometric and absolute. Ratiometric measurements measure two signals and divide them to find a relative change in intensity. For example, infrared absorption spectroscopy acquires a spectroscopic signal when nosample is present, this is the reference signal and then acquires a spectroscopic signal when a sample is present. The ratio of these signals is independent of the optical throughput characteristics of the spectroscopic system and is independent of the intensity of the optical source. Due to the universality of the ratio (absorbance) this method is very popular for quantitative measurements. The spectrum acquired when infrared light is the source is a vibrational spectrum of the sample.

[0073] Absolute measurements require only one spectroscopic signal to be acquired. An example of an absolute measurement is Raman spectroscopy. In this example, a laser source illuminates the sample, and the Raman scattered light is collected to create the vibrational spectrum. This type of measurement is dependent on the intensity of the light source and of the optical throughput of the spectroscopic system. For this reason, Raman spectroscopy is not usually considered an optimal method for quantitative measurements.

[0074] This problem with absolute measurements can sometimes be corrected if the sample contains a material that is constant in concentration and if the sample contains an analyte that is changing in concentration. In this case, the constant concentration material’s spectral features can be used as an internal standard and the ratio of the analytes spectral features are reported as the ratio of the analyte to the internal reference.Application of Internal Standards to the Problem of Antibiotic Susceptibility Tests (ATSs) Using SERS Spectroscopy

[0075] A basic workflow of an example AST process is illustrated in Figure 10. Figure 10 shows an example workflow to create a SERS AST with a blood serum infection (bacteremia or septicemia), urinary tract infection (UTI), Bronchoalveolar Lavage (BAL), and Cerebral Spinal Fluid (CSF). After a conventional sample collection and preparation, a plurality of sample containers containing nutrient broth with an increasing concentration of an antibiotic is allowed to incubate for a period of time. In this example, the process has a demonstration for the process with a 10-minute incubation. For this method each of the plurality of samples is filtered with a passive filter, such as a filter with an absorptive pad placed under a filter with a pore size sufficiently small to filter the pathogen from a broth / antibiotic mixture. This step is followed by a wash with distilled water to remove residual broth / antibiotic and to create a nutrient free environment around the filtered pathogens. When bacteria or fungi are placed in a nutrient free environment, they undergo a starvation response under which they degrade their RNA to produce energy and purines andpyrimidines as waste. The purines in particular bind strongly through a chemisorption process to SERS active nanoparticles. In order to see the purines produced in the next step, SERS active nanoparticles are added as a colloidal solution to the container with the filtered and starved pathogens. At this point the RNA’s purines and pyrimidines attach to the SERS active nanoparticles to produce a Raman signal that correlates to their concentration. A Raman spectrum can be acquired and analyzed to show if these materials are present.

[0076] Figure 11 illustrates an example filtering step to remove broth from the samples. In this example, a plate of wells is used to measure a pathogen against a plurality of antibiotics grown in a Mueller Hinton Broth (MHB). The containers in the case are represented as wells as might be found on a conventional 96 well plate. The well comprises an absorption pad under a 0.45 micron nylon filter. These are pressed against the bottom of the well with a knife edge structure to contain the flow through the filter and laterally outside of the well boundaries.

[0077] Figure 12 illustrates example steps of washing the pathogens with distilled water to initiate a starvation response and the addition of SERS active nanoparticles to the well. In this example, the SERS active nanoparticles do not pass through the filter, they sit on top of the filter and / or within the pores of the filter. The SERS-active nanoparticles pair with the purine and / or pyrimidine species to create a SERS signal. In this example, gold nanoparticles are used as the SERS-active nanoparticles.

[0078] Figure 13 shows the raw data collected from the process shown in Figure 10. The bacteria chosen for this experiment is E. Coli KI 2 and the antibiotic is Metronidazole. E. Coli KI 2 is known to be susceptible to Metronidazole at a Minimum Inhibitory Concentration (MIC) above 64 mg / L. The raw data demonstrates the problem with absolute measurements. While the laser power used to acquire these data was constant, variations in the laser focus and fluorescence from the bacteria caused the data to have unequal baselines making the peak height a poor predictor of the intensity of the analyte peak, and thus was not predictive of the MIC.

[0079] Figure 14 shows data from Figure 4 processed with a nylon reference peak. This set of data clearly shows when the E. Coli KJ 2 becomes susceptible to the concentration of the Metronidazole and the plot of the ratio of the adenine peak to a nylon peak shows the dramatic effect of the antibiotic’s shutdown of the starvation response when its concentration is above theMIC. The application of the filter’s Raman peak intensities creates a perfect internal standard for the determination of the MIC by a ratiometric measurement.

[0080] Medical devices, such as the proposed SERS AST, may, in some instances, require proof of calibration for FDA approval. Figure 14 demonstrates how the nylon filter material can be used to ensure proper calibration of the intensities and frequencies.

[0081] Figure 15 shows a plurality of potential intensity reference peaks that could be used for calibration (e.g., all of the potential intensity reference peaks).

[0082] In addition to intensity calibration spectroscopic systems can also provide a calibration for their x-axis, in the case wavenumbers. This is typically done using various chemicals with accepted peaks and their wavenumber locations. ASTM 1840 (American Society for Testing and Materials) and outlines how this can be performed with 8 different chemicals. The ASTM method was developed using FT-Raman as it produces accurate calibrations through the Connes’ Advantage.

[0083] Figure 15 shows the intensities of the nylon peaks produced by an FT-Raman spectrometer. While an FT-Raman spectrometer may be used for a SERS-AST measurement, higher sensitivity might be achievable with a dispersive spectrometer. The disadvantage of dispersive spectrometers is that they are subject to calibration loss due to laser instability, thermal effects in the alignment, and variations in alignment of the optical path. For this reason, dispersive Raman systems usually require periodic calibration (usually with one of the ASTM 1840 materials). In addition to intensity standards, Figure 15 (acquired with an FT-Raman spectrometer) provides a frequency calibration with every spectrum acquired on a nylon filter. This is an enabling improvement over periodic external calibration with ASTM 1840 standards.

[0084] While the examples have involved nylon as the filter material, these filters for pathogen filtration are available in many different materials. Figure 16 illustrates just a few examples and a SERS spectrum of adenine (the dominant purine in SERS AST measurements). All of these materials filter the pathogens well, but this set of examples demonstrates the importance of selecting the right filter. The PTFE filter material has a Raman feature almost exactly at the same location as the adenine peak thus it would be a poor choice for a filter in an application targeting adenine.

[0085] In addition to the intensity of the analyte peaks, it can also be useful to use the peak locations. Figure 17 shows the spectra of adenine produced by starved E. Colt KI 2. The peakappears at the location for adenine (731.8 cm'1). Whereas MHB also has a peak due to an purine specie (NADH, ATP, Hypoxanthine Xanthine, Uric Acid or others) that are present in the broth. MHB is the broth specified by the CLSI (Clinical and Laboratory Standards Institute). Observing the location of the analyte peak could determine if it is due to the starvation response of the pathogen (731.8 cm'1) or due to residual MHB (733.6 cm'1). This represents another example of the importance of internal frequency calibration provided by the filter material.

[0086] Figure 18 illustrates a spectrometer 100 configured to take a spectroscopic reading of a a sample adsorbed to a filter of a single well 110 (such as shown in Figure 5) and / or a plurality of spectroscopic readings of samples adsorbed to a plurality of filters of a multi-well plate (such as shown in Figure 8).

Claims

CLAIMSWhat is claimed is:

1. A method comprising: providing a solution comprising bacteria incubated in a media to a well; filtering the solution via adsorption to trap at least a portion of the bacteria in a filter separate from the media; providing a solution of SERS active nanoparticles to the well; and spectroscopically analyzing the filter to determine the presence and / or absence of at least one of a purine and a pyrimidine.

2. The method of claim 1, wherein the operation of filtering the solution comprises at least one wash of the bacteria solution through the filter.

3. The method of claim 2, wherein the operation of washing the solution comprises washing the solution with distilled water to initiate a starvation response in the bacteria.

4. The method of claim 2, wherein the operation of filtering the solution comprises passively filtering the solution through the filter.

5. The method of claim 1, wherein the filter lacks a spectroscopic feature in common with at least one spectroscopic feature of the at least one of the purine and the pyrimidine.

6. The method of claim 1, wherein the media lacks a spectroscopic feature in common with at least one spectroscopic feature of the at least one of the purine and the pyrimidine.

7. The method of claim 1, wherein the media comprises a purine-free media.

8. The method of claim 7, wherein the media comprises casein hydrolysate.

9. The method of claim 1, wherein the solution comprises different concentrations of at least one antibiotic.

10. The method of claim 1, wherein the filter is a purine-free filter.

11. The method of claim 1, wherein the filter lacks a spectroscopic signal peak corresponding to at least one spectroscopic signal peak of the at least one of the purine and the pyrimidine.

12. The method of claim 1, wherein the well is disposed adjacent to the filter.

13. The method of claim 12, wherein the filter is disposed within a mechanical barrier to prevent lateral flow of the bacterial solution and / or a wash fluid.

14. The method of claim 1, further comprising identifying the bacteria based upon an intensity of the spectroscopic analysis of the at least one of the purine and the pyrimidine.

15. A spectroscopic assay device comprising: a plurality of wells; a plurality of adsorptive filters each disposed adjacent to one of the plurality of wells; and a plurality of mechanical barriers each surrounding one of the plurality of filters and configured to prevent lateral flow of a bacterial solution and / or a wash fluid outside of each of the plurality of filters.

15. The spectroscopic assay device of claim 14, wherein each of the plurality of adsorptive the filters lacks a spectroscopic feature in common with at least one spectroscopic feature of the at least one of the purine and the pyrimidine.

16. The spectroscopic assay device of claim 14, wherein the filter is a purine-free filter.

17. The spectroscopic assay device of claim 14, wherein the filter lacks a spectroscopic signal peak corresponding to at least one spectroscopic signal peak of the at least one of the purine and the pyrimidine.

18. A spectroscopic assay device comprising: a well plate defining a plurality of wells each comprising a first inlet opening and a second outlet opening; at least one adsorptive filter comprising a first inlet side and a second outlet side, the first inlet side disposed across each of the plurality of second outlet openings of the plurality of wells; and a bottom plate disposed opposing the second outlet opening of each of the plurality of wells and at least partially enclosing the adsorptive filter between the well plate and the bottom plate.

19. The spectroscopic assay device of claim 18, wherein at least one of the bottom plate and the well plate comprise at least one mechanical barrier, such as a protrusion, projection, or knife- edge, pinching a portion of the adsorptive filter between the bottom plate and the well plate.

20. The spectroscopic assay device of claim 19, wherein the at least one mechanical barrier reduces flow within the adsorptive filter past the at least one mechanical barrier.

21. The spectroscopic assay device of claim 19, wherein the at least one mechanical barrier extends around a perimeter of the well to reduce flow within the adsorptive filter beyond the at least one mechanical barrier.

22. The spectroscopic assay device of claim 18, wherein the adsorptive filter comprises a purine free filter.

23. The spectroscopic assay device of claim 18, wherein the adsorptive filter lacks a spectroscopic feature in common with at least one of a purine and a pyrimidine.

24. The method or spectroscopic assay device of any of the preceding claims wherein an intensity of a reference peak of a component of a spectroscopic assay device, such as a filter, well, well plate, structure, or well is used as an internal standard.

25. The method or spectroscopic assay device of any of the preceding claims wherein a wavenumber of a spectroscopic reading of a component of a spectroscopic assay device, such as a filter, well, well plate, structure, or well is used as an internal standard.

26. The method or spectroscopic assay device of any of the preceding claims wherein a spectroscopic peak of a component of a spectroscopic assay device, such as a filter, well, well plate, structure, or well is used as an internal standard.

27. The method or spectroscopic assay device of any of the preceding claims, wherein a component of a spectroscopic assay device, such as a filter, well, well plate, structure, or well, provides a consistent measurement signal proportional to a spectroscopic source that is used to ratio a concentration dependent signal as an internal standard.

28. The method or spectroscopic assay device of claim 27, wherein the internal standard accounts for variations in the source intensity or its distance from a collection or focusing optics.

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