Hand-held micro-raman based detection instrument and method of detection

CROSS-REFERENCE TO RELATED APPLICATIONS

0001This application claims the benefit of U.S. Provisional Application No. 61/893,095, filed on Aug. 7, 2013. The entire disclosure of the above application is incorporated herein by reference.

FIELD

0002The present disclosure relates to a method and apparatus for rapidly detecting and identifying protein-based compounds including bacteria, virus, drugs, or tissue abnormalities, and more particularly a portable Raman spectroscopy based spectroscope which is adaptable for examining mucosal surfaces (nares, oral, ear), interrogating a wound site and/or inspection of a potentially contaminated object or surface for protein-based compounds including MRSA or other pathogens. The device can be adapted to interrogate tissue specimens, stool, urine, serum or secretions.

BACKGROUND

0003This section provides background information related to the present disclosure which is not necessarily prior art.

0004Methicillin resistance of <i>Staphylococcus aureus </i>(MRSA) is determined by the mecA gene which is carried by a mobile genetic element, designated staphylococcal cassette chromosome mec (SCCmec). MecA encodes a beta-lactam-resistant penicillin-binding protein called PBP2a (or PBP2′). Beta-lactam antibiotics normally bind to PBPs in the cell wall disrupting the synthesis of the peptidoglycan layer which results in bacterium death. However, since the beta-lactam antibiotics cannot bind to PBP2a, synthesis of the peptidoglycan layer and the cell wall continues. While the mechanism responsible for mecA transfer is still obscure, evidence supports horizontal transfer of the mecA gene between different staphylococcal species. Typically MRSA is diagnosed using culture based methods.

0005The Clinical and Laboratory Standards Institute (CLSI) recommends the cefoxitin disk diffusion test supplemented with the latex agglutination test for PBP2a. Phenotypic expression of resistance can vary depending on the growth conditions, as well as on the presence of subpopulations of staphylococci that may coexist (susceptible and resistant) within a culture making susceptibility testing by standard microbiological methods potentially problematic. In addition, culture takes time, usually 1 to 5 days. Faster techniques of MRSA screening by molecular methods, such as Polymerase Chain Reaction (PCR), have been developed to test for the mecA gene that confers resistance to methicillin, oxacillin, nafcillin, and dicloxacillin and other similar antibiotics. Such techniques, while faster, still take hours and are sent out to labs. In addition, commercially available molecular approaches (used for screening) are unable to detect mecA-variants of MRSA.

0006Raman spectroscopy is a reagentless, non-destructive, technique that can provide the unique spectral fingerprint of a chemical and/or molecule allowing for target identification without sample preparation. With this technique, a sample is irradiated with a specific wavelength of light whereby a small component, approximately 1 in 10⁷photons, is in-elastically scattered (at wavelengths shifted from the incident radiation). The inelastic scattering of photons, due to molecular vibrations that change the molecule's polarizability, provide chemical and structural information uniquely characteristic of the targeted substance. Raman Spectroscopy can be extremely useful in fully characterizing a material's composition, and allows for relatively fast identification of unknown materials with the use of a Raman spectral database. In addition, since Raman Spectroscopy is a non-contact and non-destructive technique, it is well suited for in-situ, in-vitro and in-vivo analysis.

0007Raman Spectroscopy has high potential for screening of bacteria, virus, drugs, as well as tissue abnormalities since it: 1) is practical for a large number of molecular species; 2) can provide rapid identification; and 3) can be used for both qualitative and quantitative analysis. A portable or handheld micro Raman based detection instruments would be useful to reliably and rapidly assess <i>Staphylococcus aureus </i>strains in wounds or nasal passages. Rapid assessment and typing would enable tracking the spread of such pathogens and could significantly decrease the number of hospital-acquired infections and the associated costs in treatment thereof.

SUMMARY

0008This section provides a general summary of the disclosure, and is not a comprehensive disclosure of its full scope or all of its features.

0009A hand-held Raman spectroscopy based device system for mucosal examination (nares, oral, ear) and wound interrogation provides a method for rapid and cost effective screening of bacteria, virus, drugs, and tissue abnormalities. The device is a nonintrusive automated near-real-time-point-of-care detection system that can enable healthcare providers to render better patient management and optimize clinical outcomes. The device includes a disposable tip element having dimensions that are small enough to fit into a small body cavity, such as the nostril. Three types of tip elements may be employed with this system—one for direct nasal interrogation, one with vacuum suction and filter, and one with proximity optics for wound interrogation. The tip element and the device enclose an assembly of optical components which allow for Raman spectral measurement that can provide the unique spectral fingerprint of a chemical and/or molecule allowing for target identification without sample preparation.

0010The device incorporates signal processing and identification algorithms for signal conditioning and target detection. Combinations of ultra-high resolution micro-filters in discrete regions (e.g., quadrants) of an area on the imaging detector array provide specific analysis of target spectral peaks. Each region or quadrant allows for discrete spectral band detection with each micro-filter providing specific wavenumber detection for spectral analysis. Discrete Raman spectral bands that distinguish a targeted substance from background interference are used to develop learning algorithms that serve as a basis for detection and target identification. By obtaining data at discrete spectral regions instead of over the entire spectral rage, acquisition time as well as spectral contributions from confounding background interference will be reduced or eliminated allowing for near-real-time assessment.

0011Methods for identification of pathogens in fluidic samples using Raman spectroscopy also form a part of the present disclosure. These methods show use a limited number of discrete spectra peaks to sample key molecular identifiers of a wide range of potential targets for specific pathogen detection. As a result, the apparatus and methods described herein can be customized for a host of target materials and implemented in a relatively small, portable form factor. In essence, an adapted system is provided, which can be readily modified to change target needs by means of a built-in learning algorithm. An exemplary learning algorithm was developed under a United States Department of Defense program for real-time pathogen detection in water as well as for real-time identification of cancer cells from tissue samples and may be adapted into a detection protocol. After pre-processing is complete, a Discriminant Function Analysis (DFA) is used to classify samples. DFA predicts membership in a group. The independent variables are the predictors and the dependent variables are the groups, based on an assumption of multivariate normality. This resulting data is used to modify the Raman-based spectral analysis executed in the hand held device.

0012Further areas of applicability will become apparent from the description provided herein. The description and specific examples in this summary are intended for purposes of illustration only and are not intended to limit the scope of the present disclosure.

DRAWINGS

0013The drawings described herein are for illustrative purposes only of selected embodiments and not all possible implementations, and are not intended to limit the scope of the present disclosure.

0014<figref idref="DRAWINGS">FIG. 1</figref> illustrates the mean spectra for various bacteria in terms of the relative intensity as a function of wavelength;

0015<figref idref="DRAWINGS">FIG. 2</figref> illustrates the wave numbers or spectral bands ascertained with DFA distinguishing the tested bacteria;

0016<figref idref="DRAWINGS">FIG. 3</figref> illustrates a cluster analysis of Raman spectra of bacteria;

0017<figref idref="DRAWINGS">FIG. 4</figref> illustrates a comparison of mean spectra for inoculated nasal swab samples (light) and MRSA 1R (dark);

0018<figref idref="DRAWINGS">FIG. 5</figref> illustrates the mean spectra of <i>Staphylococcus </i>with an expanded view of minimally obstructed spectral regions 600-740 (cm−1);

0019<figref idref="DRAWINGS">FIG. 6</figref> illustrates the mean spectra of <i>Staphylococcus </i>with an expanded view of minimally obstructed spectral regions 1200-1300 (cm−1);

0020<figref idref="DRAWINGS">FIG. 7</figref> illustrates a sample of the Raman spectra collected for three influenza virus;

0021<figref idref="DRAWINGS">FIG. 8</figref> illustrates spectral peaks for the three influenza virus at Raman shifts between 2850 and 2950 cm⁻¹;

0022<figref idref="DRAWINGS">FIG. 9</figref> illustrates spectral peaks for the three influenza virus at Raman shifts between 700 and 1700 cm⁻¹;

0023<figref idref="DRAWINGS">FIG. 10</figref> illustrates the spectra of immobilized influenza utilizing background subtraction techniques;

0024<figref idref="DRAWINGS">FIG. 11</figref> illustrates spectra of an influenza virus which has been deactivated using different deactivation procedures including UV, thermal and chemical treatment;

0025<figref idref="DRAWINGS">FIG. 12</figref> illustrates an exemplary form factor of the hand held micro-Raman based detection instrument;

0026<figref idref="DRAWINGS">FIG. 13</figref> illustrates the functional characteristics of the spectroscope shown in <figref idref="DRAWINGS">FIG. 12</figref>;

0027<figref idref="DRAWINGS">FIG. 14</figref> illustrates the components of the device shown in <figref idref="DRAWINGS">FIG. 12</figref>;

0028<figref idref="DRAWINGS">FIG. 15</figref> a disposable end effector of the device for wound and/or nasal interrogation;

0029<figref idref="DRAWINGS">FIG. 16</figref> illustrates a disposable end effector of the device with filtering function for vacuum suction application;

0030<figref idref="DRAWINGS">FIG. 17</figref> is an end view of the end effector shown in <figref idref="DRAWINGS">FIG. 16</figref>;

0031<figref idref="DRAWINGS">FIGS. 18A and 18B</figref> illustrate simple examples of the beam expander shown in <figref idref="DRAWINGS">FIG. 14</figref>;

0032<figref idref="DRAWINGS">FIG. 19</figref> illustrates the filter element shown in <figref idref="DRAWINGS">FIG. 14</figref>;

0033<figref idref="DRAWINGS">FIG. 20</figref> illustrates another embodiment of a Raman probe and detection system;

0034<figref idref="DRAWINGS">FIG. 21A-C</figref> illustrates the optical components of the Raman probe shown in <figref idref="DRAWINGS">FIG. 20</figref>;

0035<figref idref="DRAWINGS">FIG. 22A-C</figref> illustrates detailed aspects of the optical components shown in <figref idref="DRAWINGS">FIG. 20</figref>;

0036<figref idref="DRAWINGS">FIG. 23A-D</figref> illustrates a laser line filter, off-axis parabolic mirror system and hexagonal conical lens for the Raman probe shown in <figref idref="DRAWINGS">FIG. 22</figref>;

0037<figref idref="DRAWINGS">FIG. 24</figref> illustrates a portable form factor of the device shown in <figref idref="DRAWINGS">FIG. 20</figref>; and

0038<figref idref="DRAWINGS">FIG. 25</figref> is a flow chart illustrating the operating procedures carried out during a pathogen detection procedure using the hand held micro-Raman based detection instrument.

0039Corresponding reference numerals indicate corresponding parts throughout the several views of the drawings.

DETAILED DESCRIPTION

0040Through preliminary studies explained in further detail below, the feasibility of Raman Spectroscopy to assess various protein-based compounds including numerous pathogens using a laboratory Raman Spectrometer is demonstrated. For these studies the following pathogens were evaluated in the absence of background interference:

0041MSSA-1S: <i>Staphylococcus aureus </i>subsp. <i>aureus </i>(ATCC® 6538™);

0042MSSA-2S: <i>Staphylococcus aureus </i>subsp. <i>aureus </i>(ATCC® BAA1721™);

0043MRSA-1R: <i>Staphylococcus aureus </i>(ATCC® BAA1683™);

0044MRSA-2R: <i>Staphylococcus aureus </i>subsp. <i>aureus </i>(ATCC® 700787™);

0045<i>Corynebacterium </i>sp. (ATCC® 6931™);

0046<i>Staphylococcus epidermidis </i>(ATCC® 12228™);

0047Influenza APR8/34 (H1N1);

0048Influenza A/WSN/32 (H1N1); and

0049Influenza A/Udorn/72 (H2N3).

0000Preliminary studies on additional pathogens (such as <i>Bacillus subtilis, E. coli </i>K99<i>, E. coli </i>0111<i>, E. coli </i>0157<i>, Enterobacter </i>amnigenus, <i>Listeria monocytogenes, Pseudomonas aeruginosa, Rahnella aquatilis, Salmonella Schottmueller, Salmonella Typhimurium, Streptococcus pneumonia, Vibrio fluvialis</i>, and <i>Staphylococcus epidermidus</i>-ATCC#35984) demonstrate the feasibility of identification by Raman Spectroscopy as further described herein. These results provide a positive indication that a Raman spectral database for a wide variety of protein-based compounds could be developed with analysis protocols that allow for target identification and classification. As such the system and method described herein is not limited to examination, detection and identification of MRSA and/or influenza but has a broader range of application to examination, detection and identification of various pathogens, toxins and other protein-based compounds.

0050The MRSA-2R strain of <i>Staphylococcus aureus </i>has reduced susceptibility to vancomycin and was isolated from human blood from a patient with fatal bacteremia. The MSSA-2S strain of <i>Staphylococcus aureus </i>is a hyper-virulent community acquired methicillin-susceptible strain isolated in the United Kingdom. It is a complete genome sequenced strain. The MRSA 1R strain of <i>Staphylococcus aureus </i>is Methicillin resistant and was isolated from a human abscess. It is confirmed to carry the mec A gene with a SCCmec, or staphylococcal cassette chromosome mec type IV and PFGE type USA 400. The MSSA-1S strain of <i>Staphylococcus aureus </i>is Methicillin sensitive and was isolated from a human lesion [ATCC].

0051<i>Staphylococcus epidermidis </i>and <i>Corynebacterium </i>are normal flora found in the nose. <i>Staphylococcus epidermidis </i>with Corynebacteria predominantly colonizes the upper respiratory tract, especially the nostrils. <i>S. epidermidis </i>accounts for 90%-100% of the staphylococci found in the nasal cavity when <i>S. aureus </i>is not present. However, when <i>S. aureus </i>is present, the amount of <i>S. epidermidis </i>drastically decreases. Most species of <i>Corynebacterium </i>will not cause diseases in humans, however; <i>Corynebacterium diptheriae </i>NCTC 13129 is a strain that is highly infectious.

0052Samples were prepared from bacteria plated on tryptic soy agar plates. A single colony was picked and added to 5 mls of tryptic soy broth in a 10 ml culture tube. The culture tube was place on a shaker in a 37 C incubator and incubated overnight. The next day an optical density (OD) was taken to verify the consistency of the growth conditions and to provide a reference OD. The overnight culture was centrifuged at room temperature for 5 min @ 3000 rpms. After centrifuging the supernatant was removed and the bacteria pellet was resuspended with 5 mls of filtered tap water. The bacteria were centrifuged as stated and the washing process was repeated 2 more times. On the final wash the OD of the solution was measured and if the OD was greater than 1.05, water was added until an OD of 1+0.05 was obtained. 150 ul of the bacteria suspension was then placed on a UV quartz substrate (Craic technologies) for Raman spectroscopy.

0053Raman spectra were recorded with an in-via Raman microscope (Renishaw®) equipped with a 1800 l/mm grating, a 50 mW 514.5 nm laser as the excitation source at 100% laser power. The laser light was focused onto the sample though a 63× dipping objective (Leica HCX PL APO 1.2NA Corr/0.17 CS). The spectra were acquired over a spectral range of 400-3200 cm−1 with 40 accumulations at an integration time of 10 s.

0054Prior to analysis, spectra were pre-processed using: (1) derivative smoothing with a sliding window of 5; (2) range exclusion in the region of 735-874 cm−1 and 1013-1116 cm−1 to eliminate quartz dominated spectral regions; (3) background subtraction via a robust polynomial fit to remove spectral contributions due to fluorescence; and (4) vector normalization to reduce bacteria concentration effects. The mean Raman spectra are shown in <figref idref="DRAWINGS">FIG. 1</figref>.

0055A key to developing the Raman spectroscopy based detection device is the development of a Raman spectral database with analysis protocols that allow for target identification and classification. As part of the analysis protocol, Raman spectral bands that can distinguish a targeted substance from background interference are identified. These discrete bands are used to develop learning algorithms that serve as a basis for detection and identification. By obtaining data at discrete spectral regions instead of over the entire spectral range (600-1800 cm−1), acquisition time as well as spectral contributions of confounding background interference can be reduced. The spectroscopic system with discrete spectral band identification for algorithms development is detailed in embodiments of the device.

0056To identify discrete spectral bands of statistical significance, the pure spectra of bacteria in water were analyzed using discriminant function analysis, DFA (IBM SPSS Statistics 21). DFA builds a predictive model for group membership. The model is composed of discriminant functions that are based on linear combinations of predictor variables. Spectral data, that is to say wavenumber with associated Raman intensity, corresponding to the following Raman peaks were utilized: 600, 621, 643, 670, 725, 896, 935, 960, 1003, 1126, 1158, 1173, 1209, 1249, 1297, 1320, 1338, 1362, 1375, 1397, 1420, 1449, 1480, 1578, 1584, 1606, 1620, 1640, 1657 cm−1. Stepwise discriminant function analysis is used to reduce the number of variables (wavenumbers) to a subset of input into simultaneous discriminant analysis for classification. Once the model is finalized, cross validation is done based on the “leave one-out” principle in which one individual is removed from the original matrix and the discriminant analysis is then performed from the remaining observations and used to classify the omitted individual.

0057A similar procedure and analysis can be used for other pathogens such as the influenza virus described herein, as well as numerous other protein-based compounds.

0058The analysis for identifying the MRSA strains of bacteria is done based upon 2-group classification scheme. First an investigation of the ability of Raman spectroscopy to distinguish the <i>Staphylococcus </i>genus from other genus of bacteria is conducted. The <i>Staphylococcus </i>group consisted of MRSA 1R, MRSA 2R, MSSA 15, MSSA 2S, and <i>S. epidermidis</i>, while the non-<i>staphylococcus </i>group consisted of <i>Bacillus subtilis</i>, and <i>Corynebacterium </i>sp. The classification results show that 100% of cross-validated grouped cases correctly classified with 100% of the <i>Staphylococcus </i>group and 100% of the non-<i>Staphylococcus </i>group correctly classifying. Five wavenumbers were utilized in the discriminant model; 725 cm−1, 1158 cm−1, 1209 cm−1, 1420 cm−1, and 1450 cm−1, corresponding to vibrations of nucleic acids, proteins and lipids. Raman vibrational band assignments are given in Table 1 shown below.

0000<tables id="TABLE-US-00001" num="00001"><table frame="none" colsep="0" rowsep="0" pgwide="1"><tgroup align="left" colsep="0" rowsep="0" cols="3"><colspec colname="1" colwidth="42pt" align="center" /><colspec colname="2" colwidth="154pt" align="left" /><colspec colname="3" colwidth="70pt" align="left" /><thead><row><entry namest="1" nameend="3" rowsep="1">TABLE 1</entry></row><row><entry namest="1" nameend="3" align="center" rowsep="1" /></row><row><entry>Wavenumber</entry><entry /><entry /></row><row><entry>cm⁻¹</entry><entry>Tentative Assignments from Literature</entry><entry>Location</entry></row><row><entry namest="1" nameend="3" align="center" rowsep="1" /></row></thead><tbody valign="top"><row><entry>620, 640</entry><entry>Amino acids (620 cm⁻¹= phenylalanine,</entry><entry>Protein</entry></row><row><entry /><entry>640 cm⁻¹= tyrosine)</entry></row><row><entry>665-782</entry><entry>Nucleic acids (G, A, C, T, U)</entry><entry>DNA/RNA</entry></row><row><entry> 788</entry><entry>O—P—O sym str.</entry><entry>DNA</entry></row><row><entry>810-820</entry><entry>Nucleic acids (C—O—P—O—C), A-type helix</entry><entry>RNA</entry></row><row><entry>829, 852</entry><entry>Tyrosine (buried, exposed)</entry><entry>Protein</entry></row><row><entry>877-937</entry><entry>Protein [v(C—C)], carbohydrates [v(COC)],</entry><entry>Carbohydrates,</entry></row><row><entry /><entry>lipids</entry><entry>protein, lipids</entry></row><row><entry>1003</entry><entry>Phenylalanine v(C—C) ring breathing</entry><entry>Protein</entry></row><row><entry>1030-1085</entry><entry>Protein[v(C—N), v(C—C)], carbohydrate [v(C—O),</entry><entry>Protein,</entry></row><row><entry /><entry>v(C—C)], lipids</entry><entry>carbohydrate, lipids</entry></row><row><entry>1095</entry><entry>DNA: PO₂⁻ str (sym)</entry><entry>DNA</entry></row><row><entry>1126</entry><entry>Protein [(v(C—N), v(C—C)], lipids[v(C—C)],</entry><entry>Protein, lipids,</entry></row><row><entry /><entry>carbohydrates [v(C—C), v(COC) glycoside</entry><entry>carbohydrates</entry></row><row><entry /><entry>link]</entry></row><row><entry>1158</entry><entry>Protein [v(C—C)]</entry><entry>Protein</entry></row><row><entry>1175</entry><entry>Aromatic amino acids, Tyrosine [δ(C—H)],</entry><entry>Protein</entry></row><row><entry>1230-1295</entry><entry>Amide III [v(C—N), N—H bend, C═O, O═C—N</entry><entry>Protein, nucleic</entry></row><row><entry /><entry>bend], 1230 cm⁻¹= sat lipid</entry><entry>acids, lipids</entry></row><row><entry>1295, 1267</entry><entry>Lipids [δ(CH₂)] likely unsaturated</entry><entry>Lipids</entry></row><row><entry>1320-1340</entry><entry>Nucleic acids (Guanine, Adenine), proteins,</entry><entry>DNA/RNA, proteins,</entry></row><row><entry /><entry>carbs (1340 cm⁻¹)</entry><entry>carbohydrates</entry></row><row><entry>1336</entry><entry>Amino acids [C—H bend]</entry><entry>Protein</entry></row><row><entry>1375</entry><entry>Nucleic acids (T, A, G)</entry><entry>DNA</entry></row><row><entry>1420-1460</entry><entry>Lipids, carbohydrates, proteins [δ(C—H₂)</entry><entry>Lipids,</entry></row><row><entry /><entry>scissoring for each]</entry><entry>carbohydrates,</entry></row><row><entry /><entry /><entry>proteins</entry></row><row><entry>1483-1487</entry><entry>Nucleic acid (G, A), CH def.</entry><entry>DNA</entry></row><row><entry>1518-1550</entry><entry>Amide II [N—H bend, v(C—N), v(C═C)]</entry><entry>Protein</entry></row><row><entry>1575-1578</entry><entry>Nucleic acids (G, A), ring stretching</entry><entry>DNA</entry></row><row><entry>1585</entry><entry>Tryptophan, Phenylalanine</entry><entry>Protein</entry></row><row><entry>1606</entry><entry>Phenylalanine, Tyr.</entry><entry>Protein</entry></row><row><entry>1617</entry><entry>Tyrosine, Trp.</entry><entry>Protein</entry></row><row><entry>1640</entry><entry>Water</entry></row><row><entry>1650-1680</entry><entry>Amide I [v(C═O), v(C—N), N—H bend], Lipid</entry><entry>Protein, Lipid</entry></row><row><entry /><entry>[C═C str]</entry></row><row><entry>1735</entry><entry>>C═O ester str.</entry><entry>Lipids</entry></row><row><entry namest="1" nameend="3" align="center" rowsep="1" /></row></tbody></tgroup></table></tables>

0059Next, the feasibility of Raman spectroscopy to distinguish MRSA from other <i>Staphylococcus </i>species and strains is determined. The analysis continues all the way to stain identification. The DFA classification results are provided in Table 2 shown below.

0000<tables id="TABLE-US-00002" num="00002"><table frame="none" colsep="0" rowsep="0" pgwide="1"><tgroup align="left" colsep="0" rowsep="0" cols="3"><colspec colname="1" colwidth="63pt" align="left" /><colspec colname="2" colwidth="84pt" align="left" /><colspec colname="3" colwidth="119pt" align="left" /><thead><row><entry namest="1" nameend="3" rowsep="1">TABLE 2</entry></row><row><entry namest="1" nameend="3" align="center" rowsep="1" /></row><row><entry>Groups</entry><entry>Cross validated Results</entry><entry>Wavenumbers for the DF</entry></row><row><entry namest="1" nameend="3" align="center" rowsep="1" /></row></thead><tbody valign="top"><row><entry /></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="266pt" align="center" /><tbody valign="top"><row><entry>Genus</entry></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="3"><colspec colname="1" colwidth="63pt" align="left" /><colspec colname="2" colwidth="84pt" align="left" /><colspec colname="3" colwidth="119pt" align="left" /><tbody valign="top"><row><entry>Group 1:</entry><entry>100% of cross-validated</entry><entry>5 wavenumbers</entry></row><row><entry><i>Staphylococcus</i></entry><entry>grouped cases correctly</entry><entry>Nucleic acids (725 cm⁻¹), Protein</entry></row><row><entry>Group 2: <i>Bacillus</i></entry><entry>classified with 100%</entry><entry>(1158 cm⁻¹, 1209 cm⁻¹),</entry></row><row><entry>and Cory</entry><entry><i>Staphlocollus </i>and 100%</entry><entry>Lipids/protein (1420 cm⁻¹),</entry></row><row><entry /><entry>(Cory and <i>Bacillus</i>)</entry><entry>Lipids/protein/carbohydrates</entry></row><row><entry /><entry>correctly classifying.</entry><entry>(1450 cm⁻¹)</entry></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="266pt" align="center" /><tbody valign="top"><row><entry>MRSA from other staph</entry></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="3"><colspec colname="1" colwidth="63pt" align="left" /><colspec colname="2" colwidth="84pt" align="left" /><colspec colname="3" colwidth="119pt" align="left" /><tbody valign="top"><row><entry>Group 1:</entry><entry>90.7% of cross-validated</entry><entry>6 wavenumbers</entry></row><row><entry>MRSA1R and</entry><entry>grouped cases correctly</entry><entry>Protein (621 cm⁻¹, 1173 cm⁻¹,</entry></row><row><entry>MRSA 2R</entry><entry>classified with 89.9%</entry><entry>1338 cm⁻¹). Protein, lipids,</entry></row><row><entry>Group 2: MSSA</entry><entry>MRSA and 91.3% (MSSA</entry><entry>carbohydrates (1126 cm−¹), Lipid</entry></row><row><entry>1S, MSSA 2S,</entry><entry>and <i>S. epidermidis</i>)</entry><entry>(1297 cm⁻¹), Lipids/protein (1420 cm⁻¹)</entry></row><row><entry>and <i>S. epidermidis</i></entry><entry>correctly classifying.</entry></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="266pt" align="center" /><tbody valign="top"><row><entry>MRSA 1R vs MRSA 2R</entry></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="3"><colspec colname="1" colwidth="63pt" align="left" /><colspec colname="2" colwidth="84pt" align="left" /><colspec colname="3" colwidth="119pt" align="left" /><tbody valign="top"><row><entry>Group 1: MRSA</entry><entry>100% of cross-validated</entry><entry>3 wavenumbers</entry></row><row><entry>1R</entry><entry>grouped cases correctly</entry><entry>Nucleic acids (1320 cm⁻¹, 1584 cm−¹),</entry></row><row><entry>Group 2: MRSA</entry><entry>classified with 100%</entry><entry>Lipids/protein/carbohydrates</entry></row><row><entry>2R</entry><entry>MRSA 1R and 100%</entry><entry>(1375 cm⁻¹)</entry></row><row><entry /><entry>MRSA 2R correctly</entry></row><row><entry /><entry>classifying.</entry></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="266pt" align="center" /><tbody valign="top"><row><entry>MSSA from <i>S. epidermidis</i></entry></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="3"><colspec colname="1" colwidth="63pt" align="left" /><colspec colname="2" colwidth="84pt" align="left" /><colspec colname="3" colwidth="119pt" align="left" /><tbody valign="top"><row><entry>Group 1: MSSA</entry><entry>93.8% of cross-validated</entry><entry>5 wavenumbers</entry></row><row><entry>1S and MSSA 2S</entry><entry>grouped cases correctly</entry><entry>Protein (642 cm⁻¹, 1338, cm⁻¹).</entry></row><row><entry>Group 2:</entry><entry>classified with 93.9%</entry><entry>Protein, lipids, carbohydrates</entry></row><row><entry><i>S. epidermidis</i></entry><entry>Staphlocollus and 93.5%</entry><entry>(1126 cm⁻¹, 1450 cm⁻¹), Nucleic</entry></row><row><entry /><entry>(Cory and <i>Bacillus</i>)</entry><entry>acids (1578 cm⁻¹)</entry></row><row><entry /><entry>correctly classifying.</entry></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="266pt" align="center" /><tbody valign="top"><row><entry>MSSA 1S from MSSA 2S</entry></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="3"><colspec colname="1" colwidth="63pt" align="left" /><colspec colname="2" colwidth="84pt" align="left" /><colspec colname="3" colwidth="119pt" align="left" /><tbody valign="top"><row><entry>Group 1: MSSA</entry><entry>100% of cross-validated</entry><entry>2 wavenumbers</entry></row><row><entry>1S</entry><entry>grouped cases correctly</entry><entry>Lipid/protein (1420 cm⁻¹),</entry></row><row><entry>Group 2: MSSA</entry><entry>classified with 100%</entry><entry>Lipids/protein/carbohydrates</entry></row><row><entry>2S</entry><entry>MSSA1S and 100%</entry><entry>(1450 cm⁻¹)</entry></row><row><entry /><entry>MSSA 2S correctly</entry></row><row><entry /><entry>classifying.</entry></row><row><entry namest="1" nameend="3" align="center" rowsep="1" /></row></tbody></tgroup></table></tables>

0060The first column in Table 2 lists members of each group. The second column lists the cross-validated classification results. The third column lists the specific wavenumbers utilized in the Discriminant function models which are shown accumulatively in <figref idref="DRAWINGS">FIG. 4</figref>. The results of this analysis indicate that MRSA can be separated from other bacteria down to the strain level using a minimal number of Raman spectral bands. Further, methacillin sensitive strains of bacteria can also be distinguished and identified.

0061Next, the detection of a target pathogen with a confounding background is considered. For nasal analysis, background interference from potential confounding factors is assessed. <i>S. aureus </i>most commonly colonizes the anterior nares (the nostrils), although the respiratory tract, opened wounds, intravenous catheters, and urinary tract are also potential sites for infection. Since there are other bacteria and material in the exterior nares, it is important to investigate the ability to separate MRSA from other species of bacterium and confounding factors. Prominent nasal flora include <i>Staphylococcus aureus, Staphylococcus epidermidis </i>cells, <i>Corynebacterium </i>sp., and <i>Propionibacterium </i>sp. Nasal secretions may also include Mucin, Epithelial Cells and red blood cells.

0062For this work nasal swab samples are taken and inoculated with MRSA 1R, MRSA 2R, MSSA 1S or <i>S. epidermidis</i>. To determine if MRSA can be distinguished in the presence of nasal secretions, a cluster analysis is performed. The pure spectra, of <i>Corynebacterium </i>sp., <i>Staphylococcus epidermidis</i>, MSSA 2S and MRSA 2R in water as well as the spectra of nasal swab samples inoculated with MRSA 2R are analyzed. <figref idref="DRAWINGS">FIG. 5</figref> show the results of a cluster analysis. The results show that nasal swab samples inoculated with MRSA 2R are grouping with pure samples of MRSA 2R indicating that Raman spectroscopy can be used to distinguish bacteria in the presence of confounding factors.

0063To determine regions of the Raman spectra that are not dominated by background interference, the pure spectra of MRSA 1R (in the absence of background factors) was overlaid on the spectra of inoculated nasal swab samples. <figref idref="DRAWINGS">FIG. 6</figref> indicate that regions around 640-740, 1200-1265, 1520-1560 and 1620-1700 cm−1 have minimal background contribution.

0064The spectra shown in <figref idref="DRAWINGS">FIG. 6</figref> are pre-processed slightly different than those shown in previous figures. Due to the large intense peaks of background components, spectra were pre-processed with (1) derivative smoothing using a sliding window of 5; (2) background subtraction via a robust polynomial fit to remove spectral contributions due to fluorescence; and (3) normalization using the 1657 cm−1 peak as opposed to vector normalization.

0065The pure spectra, of bacteria in water, were re-analyzed with DFA using data only in the regions 640-740, 1200-1265 cm⁻¹. The ability of Raman spectroscopy to distinguish the <i>Staphylococcus </i>genus from other bacteria genus is shown in below. Five wavenumbers are utilized in the discriminant model; 640 cm⁻¹, 672 cm⁻¹, 725 cm⁻¹, 1209 cm⁻¹, and 1225 cm⁻¹, corresponding to vibrations of nucleic acids, and proteins.

0000<tables id="TABLE-US-00003" num="00003"><table frame="none" colsep="0" rowsep="0"><tgroup align="left" colsep="0" rowsep="0" cols="3"><colspec colname="1" colwidth="49pt" align="left" /><colspec colname="2" colwidth="84pt" align="left" /><colspec colname="3" colwidth="84pt" align="left" /><thead><row><entry namest="1" nameend="3" rowsep="1">TABLE 3</entry></row><row><entry namest="1" nameend="3" align="center" rowsep="1" /></row><row><entry>Groups</entry><entry>Cross-validated Results</entry><entry>Wavenumbers for the DF</entry></row><row><entry namest="1" nameend="3" align="center" rowsep="1" /></row></thead><tbody valign="top"><row><entry>Genus</entry><entry /><entry /></row><row><entry>Group 1:</entry><entry>94.5% of cross-validated</entry><entry>5 wavenumbers</entry></row><row><entry><i>Staphylococcus</i></entry><entry>grouped cases correctly</entry><entry>Protein (640 cm⁻¹,</entry></row><row><entry>Group 2:</entry><entry>classified with 95.8%</entry><entry>1209 cm⁻¹), Nucleic acids</entry></row><row><entry><i>Bacillus</i></entry><entry><i>Staphylococcus </i>and 91.3%</entry><entry>(672 cm⁻¹, 725 cm⁻¹),</entry></row><row><entry>and Cory</entry><entry>(Cory and <i>Bacillus</i>)</entry><entry>edge of Amide III</entry></row><row><entry /><entry>correctly classifying.</entry><entry>peak (1225 cm⁻¹)</entry></row><row><entry namest="1" nameend="3" align="center" rowsep="1" /></row></tbody></tgroup></table></tables>

0066The classification results show that 94.5% of cross-validated grouped cases correctly classified with 95.8% of the <i>Staphylococcus </i>group, and 91.3% of the non-<i>Staphylococcus </i>group (Cory and <i>Bacillus</i>) correctly classifying. These results indicate that the regions of 640-740 cm⁻¹, 1200-1265 cm−1 have potential for bacteria identification. To test the model further, nasal swab samples inoculated with MRSA 1R, MRSA 2R, MSSA 1S or <i>S. epidermidis </i>were input into the analysis as unknowns. 100% of the cases correctly classified as <i>staphylococcus</i>. Further, the mean spectra of the <i>Staphylococcus </i>species and strains show clear distinction in these regions as best seen in <figref idref="DRAWINGS">FIGS. 8 and 9</figref>.

0067The preliminary studies detailed above have shown a high confidence level that staph in general can be identified with 5 or less Raman spectral regions. In one embodiment of the invention, the system is designed to acquire Raman measurements in the presence of confounding factors. Measurements will be made directly in the nasal vestibule. The spectral regions of 640-740 cm−1, 1200-1265 cm−1, 1640-1740 cm−1 have minimal spectral components due to confounding factors and show utility for this application. In another embodiment, the otoscope contains a nasal aspirator allowing the sample to be drawn into the end effector of the otoscope through an internal filter. This filter in procedure will reduce the signal from background interference. The spectral bands for this configuration are shown in <figref idref="DRAWINGS">FIG. 4</figref>.

0068An analysis for identifying influenza virus is done using a similar classification scheme. Raman spectra have been obtained for several purified influenza viruses in phosphate buffer solution using hand held micro Raman spectrometer at an excitation wavelength of 514.5 nm fitted with a fluidic probe as further described herein. An excitation wavelength of 514.5 nm, resulted in a significant fluorescence signal from the samples. However, Raman peaks associated with the target viruses were sufficiently strong to be detectable from the background fluorescent signal. A sample of the Raman spectra collected for three influenza virus examined are shown in <figref idref="DRAWINGS">FIG. 7</figref>. The three strains of influenza for which preliminary results are shown are as follows: A/PR/8 and A/WSN/33 both being of the H1N1 serotype and A/Udorn/72 of the H3N2 serotype.

0069The results confirm that Raman spectra can be obtained for influenza virus. In addition to confirming the utility of Raman for the investigation of influenza viruses, the data collected confirms that a number of Raman peaks exist for identification purposes. A comparison of the Raman spectra for A/PR/8 and A/WSN/33 shows a sufficient difference in the spectra, which provides distinguishing characteristics between viruses with the same serotype. The Raman spectra of all three viruses in <figref idref="DRAWINGS">FIG. 8</figref> show a clear triplet of peaks at Raman shift between 2850 and 2950 cm⁻¹. These peaks are clearly present on all influenza viruses that we have been examined to date.

0070This sample data clearly provides virus detection in general as compared to spectrum from other biological entities. At Raman shifts of approximately 700 to 1700 cm⁻¹, as shown in <figref idref="DRAWINGS">FIG. 9</figref>, a large number of distinct peaks are observed for all virus samples. While many of these peaks are common to all viruses tested, a close examination shows that the relative heights of the specific peaks as well as shifts in the position of some peaks differ with each virus strain. It is these peak ratios and shifts that are utilized to distinguish the various strains from one another. To increase the sensitivity and data characteristics of influenza, <figref idref="DRAWINGS">FIG. 10</figref> shows the spectra of immobilized influenza utilizing background subtraction techniques. The spectral bands clearly identify the distinguishing pleated sheet structure amide I group as well as distinct carbon-carbon nucleic acids and other amide groups. These results clearly indicate influenza distinguishing abilities for Raman spectroscopy identification. The improvements in sensitivity and an increase in resolution in the described system will help in identifying and distinguishing differences in this region.

0071The present disclosure further enables an analysis for distinguishing a live (active) virus from a dead (inactivated) virus. For example, results from sampling inactivated dried samples of A/PR/8 (H1N1) serotype influenza run at an excitation wavelength of 785 nm revealed difference in the Raman spectra of the virus based on the inactivation method utilized. The present disclosure has heretofore focused on active pathogen samples; however, preliminary results of testing the apparatus and methods described herein showed the Raman spectral data could be used to deactivation effects of the virus. In order to determine the deactivation effects of the virus, a sample of A/PR/8 was deactivated by three distinct methods: UV, heat, and chemical deactivation. When these samples were examined at an excitation wavelength of 785 nm, clear difference in the Raman spectra of the sample that was chemically deactivate were observed as can be seen in <figref idref="DRAWINGS">FIG. 11</figref>. This difference is most obvious in the shift of the peak from 1080 to 1040 cm⁻¹, but can also be seen in the minor shift of the peak located near 1340 cm⁻¹. Minor difference also exists between the UV and heat deactivated influenza samples, but indicates the sensitivity to change in the analysis. This information is useful for the identifying changes or mutations in the target virus. For example, <figref idref="DRAWINGS">FIG. 7</figref> shows a significant portion of the mean Raman spectra of APR8/34 (H1N1), A/WSN/32 (H1N1), and A/Udorn/72 (H3N2) after preprocessing. Approximately 12 spectra averaged of each pathogen were averaged and the classification results are reproduced in

0000Table 4 below.

0000<tables id="TABLE-US-00004" num="00004"><table frame="none" colsep="0" rowsep="0" pgwide="1"><tgroup align="left" colsep="0" rowsep="0" cols="3"><colspec colname="offset" colwidth="133pt" align="left" /><colspec colname="1" colwidth="98pt" align="center" /><colspec colname="2" colwidth="28pt" align="center" /><thead><row><entry /><entry namest="offset" nameend="2" rowsep="1">TABLE 4</entry></row></thead><tbody valign="top"><row><entry /><entry namest="offset" nameend="2" align="center" rowsep="1" /></row><row><entry /><entry>Predicted Group</entry><entry /></row><row><entry /><entry>Membership</entry></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="5"><colspec colname="1" colwidth="133pt" align="center" /><colspec colname="2" colwidth="28pt" align="center" /><colspec colname="3" colwidth="35pt" align="center" /><colspec colname="4" colwidth="35pt" align="center" /><colspec colname="5" colwidth="28pt" align="center" /><tbody valign="top"><row><entry>Virus Type</entry><entry>WSN</entry><entry>PR-8-34</entry><entry>UDORN</entry><entry>Total</entry></row><row><entry namest="1" nameend="5" align="center" rowsep="1" /></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="8"><colspec colname="1" colwidth="28pt" align="left" /><colspec colname="2" colwidth="28pt" align="left" /><colspec colname="3" colwidth="42pt" align="left" /><colspec colname="4" colwidth="35pt" align="left" /><colspec colname="5" colwidth="28pt" align="char" char="." /><colspec colname="6" colwidth="35pt" align="char" char="." /><colspec colname="7" colwidth="35pt" align="char" char="." /><colspec colname="8" colwidth="28pt" align="char" char="." /><tbody valign="top"><row><entry>Original</entry><entry>Count</entry><entry /><entry>WSN</entry><entry>11</entry><entry>1</entry><entry>0</entry><entry>12</entry></row><row><entry /><entry /><entry>dimension 2</entry><entry>PR-8-34</entry><entry>0</entry><entry>8</entry><entry>0</entry><entry>8</entry></row><row><entry /><entry /><entry /><entry>UDORN</entry><entry>0</entry><entry>0</entry><entry>12</entry><entry>12</entry></row><row><entry /><entry>%</entry><entry /><entry>WSN</entry><entry>91.7</entry><entry>8.3</entry><entry>.0</entry><entry>100.0</entry></row><row><entry /><entry /><entry>dimension 2</entry><entry>PR-8-34</entry><entry>.0</entry><entry>100.0</entry><entry>.0</entry><entry>100.0</entry></row><row><entry /><entry /><entry /><entry>UDORN</entry><entry>.0</entry><entry>.0</entry><entry>100.0</entry><entry>100.0</entry></row><row><entry namest="1" nameend="8" align="center" rowsep="1" /></row></tbody></tgroup></table></tables>

0072Example embodiments of a hand held micro Raman based detection instrument will now be described more fully with reference to <figref idref="DRAWINGS">FIGS. 12-24</figref> of the accompanying drawings. Example embodiments are provided so that this disclosure will be thorough, and will fully convey the scope of this disclosure to those who are skilled in the art. Specific details may be set forth to provide a thorough understanding of embodiments of the present disclosure. It will be apparent to those skilled in the art that specific details need not be employed, that example embodiments may be embodied in many different forms and that neither should be construed to limit the scope of the disclosure. In some example embodiments, well-known processes, well-known structures, and well-known technologies are not described in detail.

0073The terminology used herein is for the purpose of describing particular example embodiments only and is not intended to be limiting. As used herein, the singular forms “a,” “an,” and “the” may include the plural forms as well, unless the context clearly indicates otherwise. The terms “comprises,” “comprising,” “including,” and “having,” are inclusive and therefore specify the presence of recited structure(s) or step(s); for example, the stated features, integers, steps, operations, groups elements, and/or components, but do not preclude the presence or addition of additional structure(s) or step(s) thereof. The methods, steps, processes, and operations described herein are not to be construed as necessarily requiring performance in the stated or any particular order discussed or illustrated, unless specifically identified as an order of performance. It is also to be understood that additional, alternative or equivalent steps may be employed.

0074When structure is referred to as being “on,” “engaged to,” “connected to,” or “coupled to” other structure, it may be directly or indirectly (i.e., via intervening structure) on, engaged, connected or coupled to the other structure. In contrast, when structure is referred to as being “directly on,” “directly engaged to,” “directly connected to,” or “directly coupled to” the other structure, there may be no intervening structure present. Other words used to describe the relationship between elements should be interpreted in a like fashion (e.g., “between” versus “directly between,” “adjacent” versus “directly adjacent”). As used herein, the term “and/or” includes any and all combinations of one or more of the associated referenced items.

0075Terms of degree (e.g., first, second, third) which are used herein to describe various structure or steps are not intended to be limiting. These terms are used to distinguish one structure or step from other structure or steps, and do not imply a sequence or order unless clearly indicated by the context of their usage. Thus, a first structure or step similarly may be termed a second structure or step without departing from the teachings of the example embodiments. Likewise, spatially relative terms (e.g., “inner,” “outer,” “beneath,” “below,” “lower,” “above,” “upper”) which are used herein to describe the relative special relationship of one structure or step to other structure or step(s) may encompass orientations of the device or its operation that are different than depicted in the figures. For example, if a figure is turned over, structure described as “below” or “beneath” other structure would then be oriented “above” the other structure without materially affecting its special relationship or operation. The structure may be otherwise oriented (e.g. rotated 90 degrees or at other orientations) and the spatially relative descriptors used herein interpreted accordingly.

0076With reference now to <figref idref="DRAWINGS">FIG. 12</figref>, an exemplary form factor (e.g, Tele-View® Wireless Otoscope by Advanced Monitors Corp.) for the hand held Raman spectroscopy based system <b>10</b> is shown and which includes a miniature laser package and optics. The hand held system may be configured as an otoscope for testing in ears, nose and throat, as an ophthalmoscope for testing in the eyes or, more generally, as a hand held spectroscope for testing wounds sites, food or inanimate surfaces. Functional components of the system components include a hand held form factor housing <b>12</b> and, a disposable interrogation tip or end effector <b>14</b> that is used for nasal interrogation. The system can be used with three types of end effectors—one for direct nasal interrogation, one with vacuum suction and filter, and one with proximity optics for wound interrogation. Other components not shown in <figref idref="DRAWINGS">FIG. 12</figref> but illustrated and described hereinafter include optical sampling head having a hybrid micro mirror, an integrated micro CCD or CMOS imager with ultra-high resolution narrow range spatially graded filter (takes the place of a large delicate spectrometer), signal processing and identification algorithms for signal conditioning and target detection.

0077A key to developing a hand-held Raman spectroscopy based device is the development of analysis protocols that allow for target identification and classification. Spectral analyses from discrete Raman bands that distinguish a targeted substance from background interference form the basis of this development. These discrete bands are used to develop learning algorithms that serve as a basis for detection and identification. A diagram of the functionality of hand-held device is schematically illustrated in <figref idref="DRAWINGS">FIG. 13</figref> to include a probe front end <b>16</b>, a set of micro-graded filters <b>18</b> and an imager <b>20</b>. The set of micro-graded filters are designed for filtering at the discrete band, e.g. filters <b>18</b>A-<b>18</b>D. As presently preferred, Filter <b>18</b>A is a micro-graded filter covering spectral band 640-740 cm⁻¹(˜3 nm band), filter <b>18</b>B is a micro-graded filter covering spectral band 1200-1260 cm⁻¹(˜2 nm band), filter <b>18</b>C is a micro-graded filter covering spectral band 1520-1560 cm⁻¹(˜1.3 nm band), and filter <b>18</b>D is a micro-graded filter covering spectral band 1620-1750 cm⁻¹(˜4.4 nm band). It should be noted for an excitation beam at 532 nm, filter <b>18</b>A filters a band in the range of 550.57-553.80 nm, filter <b>18</b>B filters a band in the range of 568.28-570.22 nm, filter <b>18</b>C filters a band in the range of 578.80-580.18 nm, and filter <b>18</b>D filters a band in the range of 582.17-586.61 nm. The imager <b>20</b> may be a CCD, a CMOS or other similar digital imaging devices.

0078This point-of-care (POC) diagnostic technology is relatively low cost and demonstrates feasibility for use in the resource-limited settings and triage settings to non-clinical utilities. The device allows for sample collection (with disposable nasal end effector on the device), processing and result read-out in the same area, without the need to send samples to a central collection point for processing or testing. It requires no sample manipulation and provides safe-containment of bio-hazardous material with routine disposal of the disposable tip. Output is provided in a visual format, without ambiguity, and includes a full process negative and internal positive control. Read-outs are available as inputs into medical management protocols. The system may also include an integrated barcoding system as a way of connecting a sample taken perhaps hours earlier to the individual who provided that sample.

0079As noted above, the device is designed for operation in non-ideal conditions, which are expected for a field or point of care deployable instrument. This includes an ability to operate under temperature extremes between 0 and 45 degree Celsius. If the design is such that version capable of operating from −25 to +50 degrees Celsius could be produced, but may require heaters to prevent freezing that could impact battery life. The device is also designed to be water and dirt resistant to allow the devices to operate under non-ideal conditions. Only the disposable end effector is exposed to the patient, thus no sterilization or cleaning of the device will be required between uses. The exposed surfaces of the device may be fabricated with antimicrobial or bacterium-resistant material.

0080The device, as an option, may also utilize existing bar code bracelets if already assigned at the point-of-care facility or site. The device includes a small low-power processor to operate the device, collect and analyze data, and store results. The device includes a USB controller to allow for the downloading of data from the POC device's internal storage to external devices, as well as real time display. The device on an auxiliary monitor may also include standard wireless/cellular cards if desired. The device utilizes an externally accessible, readily swappable, rechargeable battery pack for power.

0081A schematic representation of the components of the hand-held Raman spectroscopy based device <b>10</b> is shown in <figref idref="DRAWINGS">FIG. 14</figref>. In this example, radiation from laser <b>22</b> is directed through a laser line filter <b>24</b>, which transmits laser light while suppressing ambient light, to a 45° beam splitter <b>26</b>. The beam splitter <b>26</b> reflects the laser light through the disposable end effector <b>14</b> to the samples where it interacts with the sample producing a Raman shifted signal. Light is collected from the sample at a 180-degree geometry and is transmitted through the beam splitter <b>26</b> and laser blocking filter <b>28</b>. The laser blocking filter further prevents undesired laser light from reaching the detector <b>30</b>.

0082The Raman shifted signal then impinges upon a beam expander <b>32</b> (for example a simple beam expander <b>32</b>A, <b>32</b>B as shown in <figref idref="DRAWINGS">FIGS. 18A and 18B</figref>, respectively) that increase the diameter of a collimated input beam to a larger collimated output beam. The particular configuration and shape of the beam expander optics may be changed, such as off axis parabolic reflection, to make the beam expander more efficient and easily packaged within the device. The optical signals of the output beam are converted to electrical signals by an imager <b>30</b> with ultra-high resolution narrow range spatially graded filter <b>34</b> for processing. Filter <b>34</b> preferably includes a set of micro-graded filters <b>34</b>A-<b>34</b>D as described in reference to <figref idref="DRAWINGS">FIG. 13</figref>.

0083The disposable end effector <b>14</b> schematically illustrated in <figref idref="DRAWINGS">FIG. 15</figref> is an attachment that interacts with the patient either inserted into the nasal passage or in proximity to a wound or infection situs. For direct nasal or wound interrogation, a lens <b>36</b> is integrated at tip of the end effector to allow laser light be focused onto the specimens and Raman scattered light to be collected. As shown in <figref idref="DRAWINGS">FIG. 16</figref>, a modified end effector <b>14</b>′ is used when sample filtration is required. The end effector <b>14</b>′ will connect to a vacuum source <b>38</b> allowing the sample to be drawn into the end effector body through an internal filter <b>40</b>. In one embodiment, the device may be fitted with a small vacuum pump which functions as the vacuum source <b>38</b> removes gas molecules (air) from a sealed volume, denoted by the dashed line in <figref idref="DRAWINGS">FIG. 14</figref>, in order to leave behind a partial vacuum. The vacuum will draw the sample into the end effector <b>14</b>′. Raman measurement takes place at an optical window <b>42</b> fabricated out of an optically transparent material such as quartz. The optical window <b>42</b> is located concentrically within a mesh <b>44</b> and a seal <b>46</b> formed on an end of the end effector <b>14</b>′ opposite the filter <b>40</b>. Filtering the sample will reduce the signal from background interference by trapping large debris allowing bacteria or virus to pass through for measurement.

0084The system is configured to deliver and collect light from the sample using an open beam path. Lens tubes <b>24</b>, <b>38</b> are utilized to isolate the optical path and reduce stray light.

0085The end effectors <b>14</b>, <b>14</b>′ shown are disposable specula that detachably connects to the head <b>48</b> of the device <b>10</b> with, for example a twist lock connection to allow for precise optical alignment and ease of end effector (tip) removal. The end effector connectors may be equipped with or without a focusing lens. For vacuum suction application, the connector will house a lens <b>36</b> to allow laser light be focused onto the sample and Raman scattered light to be collected. For wound and direct nasal interrogation, the lens will be absent. The specula is designed as a single use component that is detached from the device head <b>48</b> and disposed in accordance with medical waste disposal procedures.

0086For this system, the incident beam and collected signal light share a common path such that a 45° beam splitter <b>26</b> is used to reflect the laser light through the optics to the sample while efficiently transmitting the returning Raman-shifted signal light. A laser-blocking filter <b>24</b> at normal incidence is used ahead of the dispersion element <b>26</b> to completely block the undesired laser light. The diameter of a collimated input beam is increased with a beam expander <b>32</b> to a larger collimated output beam. With reference to <figref idref="DRAWINGS">FIGS. 13, 19A and 19B</figref>, a set of ultra-high resolution micro-filter quadrants <b>34</b>A-<b>34</b>D are arranged in front of the imaging detector <b>30</b> and provide specific wavenumber or spectral band filtering by the discrete waveband analysis. Each quadrant <b>34</b>A-<b>34</b>D allows for discrete spectral band detection with each micro-filter providing specific wavenumber detection for spectral analysis. The quadrants <b>34</b>A-<b>34</b>D may be arranged symmetrically about the x and y axes as shown in <figref idref="DRAWINGS">FIG. 19A</figref>, or arranged in vertical bands as shown in <figref idref="DRAWINGS">FIG. 19B</figref>. The image sensor <b>30</b> converts the optical signals, into electrical signals. The imaging sensor <b>30</b> can be an integrated CCD or CMOS or the like.

0087The unique micro optical filters provide a narrow range of spatially graded filter, which span the narrow spectral region covering a specific Raman Spectral peak or narrow region of closely neighboring peaks. Commercial graded filters do not have sufficient resolution to achieve 1 cm−1 spatial resolution. The spectral wavelength is transformed to an imaging array position/intensity reading that provides a reconstruction of the spectral peaks of interest. The method of fabrication is a graded Indium Aluminum Nitride (InAlN) alloy that can provide spectral filtering by band gap engineering at any region between 1 eV and 6 eV band gap or 1240 nm to 206 nm. A hollow cathode based low energy plasma deposition is used to deposit the nitride alloy. Deposition is controlled by a sliding substrate window coordinated with a change in Indium deposition rate creating the graded optical coating.

0088A narrow line width laser <b>22</b> packaged in a module with integral drive electronics is used for Raman excitation. The wavelength and laser power is chosen based upon the application. The laser is able to be used as an open beam source or be coupled to an optical waveguide.

0089The spectrometer subsystem includes an electronic sub-system as well as an internal lithium-ion battery pack <b>52</b> to provide power to the system and allow for field-portable use. The system <b>10</b> is powered from either its internal battery pack or via an external charger/power adapter. The device <b>10</b> may have a provision for monitoring battery life and charge status. The device <b>10</b> may be designed with a USB controller (not shown) to allow for the downloading of data from the internal storage of the point of care (POC) device to external devices as well as a real time display (not shown). In the form of a compact LCD panel. The device may also be built to accommodate standard wireless/cellular communication if desired. The spectrometer electronic subsystem <b>50</b> utilizes a dedicated micro-controller to read the spectrum measured with the imaging sensor <b>30</b>, performs the basic processing of the image data, and transmits that information to a display, PC or other similar interface. As previously noted, the device <b>10</b> may be fitted with a small vacuum <b>38</b> for pump to work in conjunction with the disposable end effector <b>14</b>′ with filter for vacuum suction application.

0090In another embodiment, an device <b>110</b> is designed as a Raman probe with optic connection to a portable detection system <b>112</b> as shown in <figref idref="DRAWINGS">FIGS. 20 and 24</figref>. The device <b>110</b> is designed to deliver laser light to the sample and collect Raman scatter. To accomplish this, the device <b>110</b> is configured with waveguides, lenses, and filters that function to transmit the Raman scatter from the sample to the detection system for spectral analysis in a manner similar to that described with respect to device <b>10</b>.

0091The detection system <b>112</b> is a portable unit approximately 24 cm×10 cm×3 cm in size (6″×4″×1″). Key components include a laser <b>114</b> optically coupled to the device <b>110</b> for Raman excitation, and a spectrograph subunit <b>114</b> optically coupled to the device <b>110</b> for the measurement of Raman radiation intensity as a function of wavelength. A spectrograph subunit <b>116</b> indicated by the dashed box in <figref idref="DRAWINGS">FIG. 20</figref> can be configured as, but is not limited to: a grating spectrometer, a prism spectrometer, or an interferometer. The detection system <b>112</b> will also incorporate a micro controller <b>118</b> for signal processing and identification algorithms for signal conditioning and target detection, as well as support a user friendly graphical display that acts as the human-machine interface. A color LCD display will have sufficient resolution to display use instructions, as well as test results in text output for go/no-go classification, and to graphically display a spectra. A simple menu structure with large pushbutton icons make operation of the device straight forward and user friendly.

0092As shown in <figref idref="DRAWINGS">FIGS. 20 and 24</figref> the spectroscope subunit <b>116</b> is configured as a Czerny-Turner spectrometer. Radiation from laser <b>114</b> is directed through a flexible optical waveguide (fiber) <b>120</b> to the device <b>110</b> and is transmitted through a laser line filter <b>122</b> and disposable end effector <b>124</b> to the samples. The light interacts with the sample producing a Raman shifted signal which is collected at 180-degree geometry. The collected light is transmitted thought a laser blocking filter <b>122</b> and coupled into a flexible optical waveguide (fiber) <b>126</b>. The laser blocking filter <b>122</b> prevents undesired laser light from reaching the detector. The Raman shifted signal is directed through the optical waveguide (fibers) <b>126</b> to the spectroscope subunit <b>116</b> of the detection system <b>112</b>. Light entering the subunit <b>116</b> is reflected off of the collimating mirror <b>128</b> and is directed onto the diffraction grating <b>130</b> which separates incident polychromatic light into constituent wavelength components. The diffracted light is directed to a focusing mirror <b>132</b> onto a detector <b>134</b> which converts optical to electrical signals for processing.

0093As presently preferred, the disposable end effector <b>124</b> is a disposable specula that interacts with the patient either inserted into the nasal passage or in proximity to a wound or infection sight. The end effector design is similar to that described in <figref idref="DRAWINGS">FIGS. 15-17</figref>.

0094Further details of the optical train for the device <b>110</b> are illustrated in <figref idref="DRAWINGS">FIGS. 21-23D</figref>. Light from the laser <b>114</b> is coupled into the excitation fibers <b>120</b><i>e </i>of the probe as shown in <figref idref="DRAWINGS">FIG. 19</figref>. As best seen in <figref idref="DRAWINGS">FIG. 22A</figref>, the excitation fibers <b>120</b><i>e </i>form part of the fiber bundle <b>120</b> which are concentrically arranged around the collection fiber <b>120</b><i>c</i>. In a preferred embodiment, the collection fiber <b>120</b><i>c </i>has a diameter approximately four times larger than the diameter of the excitation fiber <b>120</b><i>e</i>. A high rejection filter (laser line filter) <b>122</b>A at the output of these fibers is used to remove Raman bands arising from the silica core, thus allowing only the laser light to be transmitted to the sample. Hollow core Photonic crystal fibers are used as excitation fibers in order to reduce/eliminate the need for filtering.

0095Off axis parabolic mirrors <b>136</b>, located beneath the excitation fibers <b>120</b> collimate and direct the beams to a 45 degree cone lens <b>138</b>. FIG. <b>22</b>C illustrate an excitation beam transmitted from the excitation fibers <b>120</b><i>e </i>and impinging on the face of the cone lens <b>138</b>. As presently preferred, the height of the cone lens <b>138</b> is approximate twice the diameter of the excitation fiber <b>120</b><i>e </i>as best seen in <figref idref="DRAWINGS">FIG. 22B</figref>. This lens <b>138</b> has dielectric coated faces that allow the laser light to be reflected and the Raman scatter to be transmitted. In particular, the outside surface of the lens <b>138</b> is coated with a dielectric to reflect laser light and pass Stokes scattered light. The reflected laser light is directed toward the sample surface and focused with a convex lens. When the lens is absent, collimated light is output from the probe. Light scattered from a sample is collected 180 degrees relative to the direction of the laser beam. It is directed through the cone lens <b>138</b> which allows only the Raman scattered light to be coupled into the collection fiber <b>126</b>.

0096Two key elements of this design are the off axis parabolic mirror <b>136</b> system and the 45 degree cone lens <b>138</b>. As best seen in <figref idref="DRAWINGS">FIG. 23B</figref>, the off axis parabolic mirror is an annular or doughnut shaped optic that has eight conic depressions or dimples <b>140</b> on its surface. Each of the eight dimples <b>140</b> forms a 90 degree parabolic mirror with its focal point at a designated excitation fiber <b>120</b><i>e</i>. As shown in <figref idref="DRAWINGS">FIGS. 21, 22B, 22C, 23C and 23D</figref>, the cone lens <b>138</b> is a hollow hexagonal optical element whose faces are at a 45 degree angle. The lens <b>138</b> has a dielectric coating enabling it to act as a long pass filter (reflecting laser light and transmitting the Raman scatter).

0097Other features incorporation into the system includes: a strain relief boot <b>142</b> which provide strain relief to fiber cables, and exhibit a high degree of flexibility. A first connector <b>144</b> secures the excitation fiber (waveguide) of the device <b>110</b> to the laser <b>114</b>. A second connector <b>146</b> secures the Raman collection fiber (waveguide) of the device <b>110</b> to the spectrograph subunit <b>116</b>. A narrow line width laser <b>114</b> is packaged in a module with integral drive electronics for Raman excitation. The wavelength and laser power is selected based upon the application and target identification. The laser is coupled to an optical fiber or waveguide with use of a third connector.

0098The second fiber connector <b>146</b> secures the input fiber <b>126</b> (or waveguide) to the spectrograph subunit <b>116</b>. Light from the input fiber (or waveguide) enters the detection system through this connector. Behind the connector, a slit (not shown) having a dark piece of material containing a rectangular aperture may be utilized. The collimating mirror <b>128</b> focuses light entering the spectrometer portion of the detection system towards the grating <b>130</b>. Diffraction grating <b>130</b> diffracts light from the collimating mirror <b>128</b> and directs the diffracted light onto the focusing mirror <b>132</b>. The dispersive element <b>130</b> separates incident polychromatic light into constituent wavelength components and can be a grating or prism or a like. Focusing mirror <b>132</b> receives light reflected from the grating <b>130</b> and focuses the light onto the CCD Detector <b>134</b>. CCD detector <b>134</b> collects the light received from the focusing mirror <b>132</b> and converts the optical signal to a digital signal. Each pixel on the CCD Detector corresponds to the wavelength of light that strikes it, creating a digital response signal.

0099As noted above, the detection system <b>112</b> includes an internal lithium-ion battery pack (not shown) to provide power to the system and allow for field-portable use. The system can be run from either its internal battery pack or via an external charger/power adapter. The device <b>112</b> will have a provision for monitoring battery life and charge status. The detection system <b>112</b> may include an electronic sub-system which includes a PC-based processor <b>148</b>, spectrometer <b>116</b>, vacuum pump and valve controller (not shown), pressure sensors (not shown), and interlocks. PC-based processor is used to perform all of the computation and coupled to an LCD display <b>150</b> with a touch screen and/or perimeter function buttons <b>152</b> to handle menu selection. The spectrometer subsystem <b>112</b> utilizes a dedicated micro-controller <b>118</b> to read the CCD array, perform basic processing on the image data, then transmit that information to the PC using a USB or other similar interface.

0100With reference now to <figref idref="DRAWINGS">FIG. 25</figref>, a flow chart <b>210</b> illustrating the detection process is provided. In particular, a hand-held Raman spectroscopic device as described above is operated to transmit a coherent light beam from the excitation laser onto a sample. The imaging sensor detects radiation from the filtered Raman-shifted sample signal (block <b>212</b>) and generates image data representative thereof (block <b>214</b>). The image data is then analyzed (block <b>216</b>) and the spectral features at discrete spectral bands are examined to detect the presence of a target pathogen (block <b>218</b>). If no target pathogenic features are found, the device displays and/or reports a negative result for the presence of the target pathogen (block <b>220</b>).

0101If target pathogenic features are found, these features are compared with baseline Raman spectra (block <b>222</b>). Algorithms and classification coefficients are computed based on the baseline spectra (block <b>224</b>). Typing of the target pathogenic features is done in a hierarchical approach and classification is assigned as the comparison moves down the hierarchy (block <b>226</b>). If a positive database match is identified, the device displays and/or reports a positive result for the presence of the target pathogen (block <b>228</b>). If a positive database match is not identified, the probability of the target pathogen's identity or membership within a particular group of interest may be computed and displayed or reported (block <b>230</b>).

0102A robust portable Raman spectroscopy based system as detailed above has many anticipated benefits. The nonintrusive, nondestructive technique for nasal examination and wound interrogation provides rapid and cost effective screening of a wide range of protein-based compounds including bacteria, virus, drugs, and tissue abnormalities. The method requires little or no sample preparation, reducing the need for storage of consumables. In addition, the ease of use and non-contact sampling make the device a valuable tool for point of care investigations.

0103The device is a reagentless automated near real time point of care detection system that can enable healthcare providers to render better patient management and optimize clinical outcomes. Since the sensor can be developed to analyze bacteria, virus, drugs, and tissue, it can be promoted to a variety of market segments that include: primary care physicians, and drug stores.

0104The foregoing description of the embodiments has been provided for purposes of illustration and description. It is not intended to be exhaustive or to limit the disclosure. Individual elements or features of a particular embodiment are generally not limited to that particular embodiment, but, where applicable, are interchangeable and can be used in a selected embodiment, even if not specifically shown or described. The same may also be varied in many ways. Such variations are not to be regarded as a departure from the disclosure, and all such modifications are intended to be included within the scope of the disclosure.