METHODS AND COMPOSITIONS FOR DETECTING TUBERCULOSIS BACKGROUND OF THE INVENTION

CROSS-REFERENCE TO RELATED APPLICATION

The present application claims priority to United States Provisional Patent Application Number 61/732,266, filed November 30, 2012 (Atty. Dkt. No. 37182.162), the contents of which is specifically incorporated herein in its entirety by express reference thereto.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under Grant No. W81XWH-1 1- 2-016 awarded by the United States Department of Defense. The government has certain rights in the invention.

NAMES OF THE PARTIES TO A JOINT RESEARCH AGREEMENT

Not Applicable. FIELD OF THE INVENTION

The present invention relates to the fields of molecular biology and medicine. In particular, the invention provides methods and compositions for the detection of tuberculosis in a sample. In illustrative embodiments, the invention provides a label-free, rapid (~one-hour), and cost-effective high-throughput diagnostic assay that detects both pulmonary and extra-pulmonary active tuberculosis disease.

DESCRIPTION OF RELATED ART

Tuberculosis (TB), an infectious disease caused by Mycobacterium tuberculosis (M. tuberculosis), continues to be a major public health challenge, accounting for 9 million new cases and 1.5 million deaths annually worldwide (World Health Organization, Global Tuberculosis Control: WHO Report 2011). In 2010, there were more than 1 1,000 new cases of tuberculosis infection reported in the United States alone. Traditional TB testing requires subjects to receive an under-the-skin injection of a harmless protein produced by Mycobacterium tuberculosis. Two or three days later, the subject is asked to return for a "reading", during which the injection site is evaluated. If a person has ever been infected by TB or is currently battling a TB infection, the injection site will appear red and irritated— a positive reading.

M. tuberculosis culture testing (MTCT) remains the "gold standard" for the diagnosis of active TB disease, as well as the identification of drug-resistance. Unfortunately, this method generally requires 6 to 8 weeks to complete (Dunlap et al, 2000; Scarpellini et al, 2004). In addition to the significant delay in receiving results, conventional TB tests are unable to detect some types of active TB disease, such as tuberculous meningitis, which does not actively shed bacteria. Sputum smear microscopy, the primary means of tuberculosis diagnosis in most parts of the world for more than a century, requires well-trained personnel to be performed correctly, and its sensitivity (35- 80%) is significantly lower than that of mycobacterial culture. The method has a number of drawbacks, including low sensitivity (especially in HIV-positive individuals and children) and an inability to determine drug-resistance. Because conventional diagnosis of drug resistant TB relies on bacterial culture and drug susceptibility testing, both slow and cumbersome processes, during that time patients may be inappropriately treated, drug- resistant strains may continue to spread, and resistance may become amplified. The method is also unable to differentiate between drug-sensitive and drug-resistant tuberculosis strains.

To determine whether TB bacteria are resistant to antibiotic drugs, current technology require a separate test that takes 3-6 weeks to complete. Rapid and reliable diagnostic tests for active tubercular disease are thus highly desirable to minimize the morbidity and mortality of this airborne infectious disease.

ESAT-6 AND CFP-10 AS BIOMARKERS OF TUBERCULOSIS

ESAT-6 (early secretory antigenic target protein) and CFP-10 (culture filtrate protein 10) are exclusively secreted by several pathogenic mycobacterial species, including M. tuberculosis, and non-tuberculosis species (NTM, M. kansasii, M. szulgai, M. marinum, and M. riyadhense), and is consistently missing from all versions of attenuated vaccine strains (Bacillus Calmette-Guerin , BCG) and other mycobacterial species (Van Ingen et al, 2009). Thus, ESAT-6 and CFP-10 are considered excellent biomarkers for TB diagnosis.

The interferon-gamma release assays (IGRAs), immunodiagnostic assays, were developed and commercialized to detect ESAT-6-immunized T-cells in whole blood (Doherty et al, 2002). Although the IGRAs are sensitive to those who have been administered the BCG vaccine, this assay still cannot distinguish between active TB disease and remote latent TB infection (LTBI), due to the immunologic response from long-lived human memory T cells (Wu-Hsieh et al, 2001). Since actively replicating M. tuberculosis strains within the human body release ESAT-6, which triggers chronic inflammation and an immune response, ESAT-6 could be detected in a patient's bodily fluids, including sputum (pulmonary TB), serum, cerebrospinal fluids (tuberculous meningitis), and pleural effusion (tuberculous pleuritis) (Kashyap et al, 2009; Sang et al, 2012; Hoff et al, 2007; Ravn et al, 2005).

Nucleic acid and nanoparticle approaches have been developed in recent years.

However, most nucleic acid-based approaches require polymerase chain reaction (PCR) methodology for amplifying nucleic acids specific to the TB bacteria, one in particular uses a nested protocol that targets the heat shock protein 65 gene (hsp65). Most nanoparticle- based approaches merely use nanoparticles to detect antigens that are already captured (Chun, 2009; Torres-Chavolla and Alocilia, 2011). Spherical gold nanoparticles have also been employed for rapid detection of MT-specific DNA using non-PCR based protocols (Hussain et al, 2013; Tsai et al, 2013).

A need remains, however, for faster, more sensitive, and more specific methods of TB detection, including assays suitable for detecting the bacterium in patient-derived biological specimens. Different, more varied approaches are needed, including peptide- and protein-based approaches that do not rely on nanoparticle-based methodologies.

BRIEF SUMMARY OF THE INVENTION

The present invention overcomes these and other limitations inherent in the prior art by providing inventive diagnostic compositions for use in the preparation of medicaments, and in methods for the detection, diagnosis, treatment, and/or amelioration of one or more symptoms of mammalian disease. In particular, the invention provides novel, non-obvious, and useful compositions that are suitable for the detection of the causal agent of tubercular infection in mammals, and in particular, for the detection of symptoms of M. tuberculosis (MTB) cells that express an MTB-specific biomarker, such as ESAT-6 or CFP-10 proteins, peptides, or one or more proteolytic fragments thereof.

The present invention utilizes nanoporous silica chips that are constructed with a dual-layered film, engineered with properties that facilitate on-chip fractionation and digestion of samples, exclusion of the abundant proteins that normally obscure the detection of the target molecules and selective capture of the rare biomarkers from a biological sample. The diagnostic chips described herein are useful in selectively purifying low- molecular-weight (LMW) TB biomarkers, and facilitating the highly sensitive detection and quantification of such biomarkers by an analytical method such as mass spectrometry (MS).

Importantly, the invention provides label-free, highly reproducible diagnostic tests that are cost-effective, and capable of identifying active TB disease including the more dangerous multi-drug-resistant tuberculosis. The method provides rapid diagnosis of TB infections (typically within one hour from sample collection to diagnosis), and importantly, can be used to distinguish between active TB disease and latent TB infection. Use of such diagnostic chips to selectively purify LMW TB biomarkers within the nanomolar range facilitates a reproducible, cost-effective, and high-throughput (-150 sample wells in a four- inch size chip) platform, which permits highly sensitive detection and quantification of biomarkers of interest by MS.

In a first embodiment, the invention provides a method of identifying at least one pathogen-specific protein or peptide from a sample. In an overall and general sense, the method generally involves contacting the sample with a nanoporous film; and detecting the presence of the αί/zogew-specific protein or peptide, or one or more proteolytic fragment(s) thereof.

In certain embodiments, the pathogen-specific protein or peptide is specific for M. tuberculosis, and may include a contiguous amino acid sequence from an early secretory antigenic target protein (ESAT-6), a culture filtrate protein 10 (CFP-10), or one or more proteolytic fragment(s) thereof.

In certain embodiments, the pathogen-specific protein or peptide comprises, consists essentially of, or alternatively, consists of at least 8, at least 9, or at least 10 or more contiguous amino acids from any one of SEQ ID O: l, SEQ ID O:2, SEQ ID O:3,

SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:7.

Alternatively, the pathogen-specific protein or peptide may comprise, consist essentially of, or alternatively, consist of at least 11, at least 12, or at least 13 contiguous amino acids from any one of SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4,

SEQ ID NO:5, or SEQ ID NO:6.

In particular embodiments, the pathogen-specific protein or peptide may comprise, consist essentially of, or alternatively, consist of at least 14, at least 15, or at least 16 contiguous amino acids from any one of SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, or SEQ ID NO:5. And in other embodiments, the pathogen-specific protein or peptide may comprise, consist essentially of, or alternatively, consist of at least 17, or at least 18 contiguous amino acids from any one of SEQ ID NO: 1, SEQ ID O:2, SEQ ID NO:3, or SEQ ID NO:4.

Preferably, the sample is a biological sample obtained from a mammal, and more particularly is a biological sample that contains sputum, pleural effusion, cerebrospinal fluid, urine, serum, plasma, and/or whole blood obtained from a human.

In some applications, the sample may be contacted with the film neat (i.e., undiluted), or alternatively, the at least one protein or peptide within the sample may be concentrated prior to contact with the nanoporous film, using a suitable method such as salt precipitation or such like.

Preferably the nanoporous film comprises a plurality of pores, substantially having the same average diameter, into which the pathogen-specific protein or peptide is absorbed. The nanoporous film may comprise a first layer of silica film that contains a plurality of pores having an average diameter of about 3 to about 10 nm, or alternatively, may contain a first layer of silica film that contains a plurality of pores having an average diameter of about 6 to about 8 nm.

In certain embodiments, the nanoporous film may further optionally include a second layer of silica film deposited upon the first layer, to form a dual-layer film. In such applications, preferably the first layer of silica film contains a plurality of pores having a first average diameter, and the second layer of silica film contains a plurality of pores having a second average diameter that is distinct from that of the first layer.

In particular commercial applications, the second layer of silica film may contain a plurality of pores having a first average diameter that is larger than that of the plurality of pores in the first layer.

The method may also further optionally include washing the nanoporous film after contacting the film with the sample, and/or digesting the sample containing the pathogen- specific protein or peptide with a protease or a peptidase to produce one or more proteolytic fragment(s) of the pathogen-specific protein or peptide.

For detection of MTB-specific peptides, the protease is preferably trypsin. The proteolysis of the sample may be performed prior to analysis, or alternatively, upon or within the nanoporous film itself during analysis.

The method may further optionally include isolating the one or more proteolytic fragment(s) from the nanoporous film with a suitable elution buffer. The presence of the pathogen-specific protein or peptide, or the one or more proteolytic fragment(s) thereof is preferably detected by identifying at least one mass fingerprint of the protein, the peptide or the proteolytic fragment(s) thereof by mass spectrometry.

In the analysis of ESAT-6 or CFP-10, the at least one mass fingerprint is identified at about 1895-1910 Da ([M+H] ⁺ ) or about 2003-2005 Da ([M+H] ⁺ ), or alternatively at about 1900.9511 Da ([M+H] ⁺ ) or 1907.9246 Da ([M+H] ⁺ ), or alternatively at about 2003.9781 Da ([M+H] ⁺ ), about 1668.7170 Da ([M+H] ⁺ ), about 1593.7503 Da ([M+H] ⁺ ), about 1142.6276 Da ([M+H] ⁺ ), or about 908.4584 Da ([M+H] ⁺ ). The at least one mass fingerprint may be detected by matrix-assisted laser desorption/ionization time-of- flight mass spectrometry (MALDI-TOF MS).

In other aspects, the invention provides a method that generally includes the steps of a) enriching at least one target protein from a sample by contacting the sample with a nanoporous film under conditions to absorb the target protein to the film, and subsequently washing the nanoporous film to remove extraneous material; b) digesting the enriched target protein on the nanoporous film to produce at least one digestion product comprising at least one proteolytic fragment thereof; and c) detecting the presence of the at least one proteolytic fragment of the target protein. In the practice of the method, at least two different target proteins, either or both of which is specific for a particular pathogen, may be from a single sample, or alternatively, may be from multiple samples.

In certain embodiments, the nanoporous film may include a silica film that contains a plurality of pores having an average diameter of about 3 to about 10 nm, and as noted above, may also include dual- or multi-layer silica films each having substantially same, or substantially different average pore sizes contained therewith to provide differential sorting of the proteins or peptides in the sample based upon size of the pores of each layer.

In another embodiment, the invention provides a method that generally includes the steps of (a) enriching at least one ESAT-6-, CFP-10-, or IF-10-specific protein or peptide from a sample containing a first mammalian bodily fluid, by contacting the sample with a nanoporous film and washing the nanoporous film, wherein the nanoporous film comprises a plurality of pores having an average diameter of about 3 to about 10 nm; (b) digesting the enriched ESAT-6-, CFP-10-, or IF-10-specific protein or peptide in the nanoporous film with at least one protease to produce a digestion product comprising at least one proteolytic fragment of an ESAT-6-, CFP-10-, or IF-10-specific protein or peptide; (c) eluting the digestion product from the nanoporous film using a biological buffer; and (d) detecting the presence of the at least one proteolytic fragment of an ESAT-6-, CFP-10-, or IF-10-specific protein or peptide in the eluted sample via mass spectrometry.

BRIEF DESCRIPTION OF THE DRAWINGS

For promoting an understanding of the principles of the invention, reference will now be made to the embodiments, or examples, illustrated in the drawings and specific language will be used to describe the same. It will, nevertheless be understood that no limitation of the scope of the invention is thereby intended. Any alterations and further modifications in the described embodiments, and any further applications of the principles of the invention as described herein are contemplated as would normally occur to one of ordinary skill in the art to which the invention relates.

The following drawings form part of the present specification and are included to demonstrate certain aspects of the present invention. The invention may be better understood by reference to the following description taken in conjunction with the accompanying drawings, in which like reference numerals identify like elements, and in which:

FIG. 1A, FIG. IB, and FIG. 1C show a schematic of an experimental procedure for one embodiment. The biological fluid was fractionated by nanoporous silica film coated on the substrates, (i) The large abundant protein was excluded by 5-8 nm size pore, (ii) After extensive washing, relatively small ESAT-6/CFP-10 was retained in the nanopore. (iii) The enzyme, trypsin, digested the ESAT-6/CFP-10 into small fragments, (iv) The protein fragments were eluted in elution buffer, and then detected by mass spectrometry (FIG. 1A); fingerprint mass spectrum of CFP-10 fragment after trypsin digestion (FIG. IB (four major fragments of CFP-10, mass 1142.6276 Da and 1317.6645 Da, 1593.7503 and 2003.9781 ([M+H] ⁺ ), exhibit high signals in MALDI mass spectra); and fingerprint mass spectrum of ESAT-6 fragment after trypsin digestion (FIG. 1C (two major fragments of ESAT-6, mass 1900.951 1 Da and 1907.9246 Da ([M+H] ⁺ ), exhibit high signals in MALDI mass spectra);

FIG. 2A, FIG. 2B, FIG. 2C and FIG. 2D show the mass spectra of ESAT-6 in human serum (ESAT-6 = 80 μΜ). Spectra were zoomed to show the 1901 peak in the insets (FIG. 2A). The serum mixed with ESAT-6 was directly digested with trypsin without on-chip fractionation. High abundant proteins hinder the signal of ESAT-6 (FIG. 2B). The serum was fractionated with NSCs, and then treated with trypsin. The efficiencies of fractionation and digestion process in NSCs with different pore sizes (ESAT-6 = 80 μΜ) were determined: FIG. 2C shows the intensity of 1901 fragments of ESAT-6. The highest signal was observed in the NSCs with 6-nm pore diameter, and FIG. 2D shows the intensity of 1901 fragment normalized by the number of pore per surface area;

FIG. 3A, FIG. 3B, FIG. 3C and FIG. 3D show BET and ellipsometry was used to measure Film characteristics and dimensions (FIG. 3A). The porosity and film thickness were measured by ellipsometry. The surface area, pore volume, and pore size were determined by ₂ adsorption/desorption analysis. The details of nanopore characterization were described in Method Section. The L121+25% PPG (thin) was expected to have the same pore morphology as standard L121+25% PPG. FIG. 3B: The proportion of un- fragmented CFP-10 that was retained in the detection well after washing (40 ng of CFP-10 was applied in a 7 mm ² size well, mean ± s.d., n = 6). L121+25% PPG can isolate up to 36 ng. FIG. 3C: MALDI MS signal intensity of each CFP-10 fragment normalized to its own isotopic fragments. Recombinant CFP-10 was spiked into the culture media, which was then treated through on-chip fractionation and digestion prior to MS analysis, (mean ± s.d.; n = 5); FIG. 3D: Measuring the amount of CFP-10 fragments recovered from sample input. Recombinant CFP-10 (40 ng) was spiked into the culture medium, which was then treated with on-chip fractionation and digestion. The absolute amounts of CFP-10 fragments (1 142.63 and 1593.75 [M+H] ⁺ ) were quantified by spiking isotopic fragment into eluted samples.;

FIG. 4 shows the depth profiles of CFP-10 enriched on L121+25% PPG, as determined from the Nls spectrum collected using XPS. The line represent the exponential fit of y=y0+A ^(~x/B) +C ^(~x/D) . CFP-10 could penetrate 100 nm into the film. The inset shows representative XPS N1 S spectra of nanoporous film with and without CFP-10;

FIG. 5 shows the relative intensity of each major CFP-10 fragment to its isotopic fragment is plotted verses the input CFP-10 concentration. The isotopic ¹⁸ 0-labeled fragments were generated by trypsin digestion in H ₂ ¹⁸ 0. Isotopic CFP-10 at 42 nM of was added in equal proportion to known digested CFP-10 before spiking on MALDI MS plate. In this condition, the 1 142.63 and 1593.75 fragments show good linear relation with their respective isotopic fragments below 400 nM;

FIG. 6A, FIG. 6B, and FIG. 6C show different amounts of recombinant ESAT-6 in urine (mean ± s.d., n = 6), and recombinant CFP-10 in MTB culture media (mean ± s.d.; n = 5). FIG. 6A: The red line represent the fit of y = ax/(l+ax). The semi-log plot was presented in the inset. The 1901 fragment was detectable when ESAT-6 concentration was above 60 nM. The detection threshold for CFP-10 fragments by MALDI-TOF MS analysis. The signals of each fragment were normalized by its own isotope as an internal standard. FIG. 6B: Un-precipitated culture medium for each CFP-10 dilution was processed through on-chip fractionation and digestion. The sensitivity plot maintained good linear regression above 13.4 nM in log-log scale. FIG. 6C: the samples were precipitated lOx by ammonium sulfate prior to on-chip processing. MS analysis showed the detection limit had been lowered to 1.3 nM because of sample concentration;

FIG. 7 shows the mass spectra of MTB-specific CFP-10 fragments. None of these fragments was observed in the culture of non-TB specie of mycobacteria (Mycobacterium avium);

FIG. 8 shows the intensity of 1901 fragment obtained by MALDI mass spectrum at different ESAT-6 concentration in human serum (mean ± s.d., n = 6). The red line represents the fit of y = ax/(l+ax). The semi-log plot is presented in the inset. The 1901 fragment was detectable when the ESAT-6 concentration was above 60 nM;

FIG. 9 shows the fingerprint mass-spectrum of full-length recombinant ESAT-6 and full-length recombinant CFP-10 collected in linear mode of MALDI TOF MS at 5 μΜ concentration. The molecular weights of recombinant ESAT-6 and recombinant CFP-10 were 13 kDa and 1 1 kDa, respectively;

FIG. 10 shows the MALDI mass spectrum of human serum treated with on-chip fractionation and trypsin digestion. No peak was selected in the range from 1895 to 1910 (Signal-to-noise ratio threshold = 3, noise-window-width=250 in Data Explorer software). The fragment from human serum did not overlap with ESAT-6 fragments at 1900.9511 Da ([M+H] ⁺ );

FIG. 11 shows the XPS depth profiles of ESAT-6 enriched in nanopores of 6- and 8-nm. The amount of ESAT-6 was determined from Nls spectra collected by XPS. The lines represented the exponential fit of y = y0+A ^(~x/B) . ESAT-6 penetrated deeper in the 8-nm nanopore because of the slower decay of depth profile (B = 29 and 48 nm for 6- and 8-nm nanopore, respectively). The total amount of ESAT-6 trapped in the 6-nm pore was higher than 8-nm NSC because there were more nanopores per surface area in 6-nm NSC. The inset represents the depth profiles normalized to the number of pore per surface area. After normalization, more ESAT-6 antigens were trapped in 8-nm NSC;

FIG. 12 shows the improvement of ESAT-6 signal with a pre-concentration procedure. 1 mL of 40 nM ESAT-6 in urine was concentrated by ammonium sulfate precipitation procedure. The precipitated proteins were dissolved in a final volume of 20 μϊ _^ buffer. Comparing to non-concentrated sample (250 nM ESAT-6), the precipitation procedure significantly improve the signal of ESAT-6; FIG. 13 shows the indirect ELISA standard curve of ESAT-6 in IX PBS, urine, and 5% diluted human serum. Indirect ELISA could reach a higher sensitivity in the samples with low background proteins. In IX PBS solution, an ESAT-6 signal was observed at 2 nM concentration. In 100% human serum, signals below micromolar concentration could not be observed;

FIG. 14 shows the fingerprinting spectra of ESAT-6 and CFP-10 fragments. Two major fragments of ESAT-6 were observed in MALDI TOF MS. Compared to ESAT-6, five fragments of CFP-10 were observed;

FIG. 15 shows the spectrum of human serum containing ESAT-6 and CFP-10 antigens after treated with on-chip fractionation. Both CFP-10 and ESAT-6 fragments could be observed simultaneously. CFP-10 showed higher intensity than ESAT-6 in MALDI TOF MS;

FIG. 16 shows the dual-layer NSC photo collected from scanning electron microscope. The top layer of the nanoporous film was made by L121+25%PPG of ~90 nm thickness, and the bottom layer was made by L121 with ~700-nm thickness; and

FIG. 17A and FIG. 17B show the spectra of human serum containing ESAT-6 and CFP-10 antigens as low as 100 nM after treated with ultracentrifugation coupling with on- chip fractionation. Two CFP-10 fragments with m/z 1593.756 and 2003.989 showed clear signals in MALDI TOF MS after treated with single-layer NSC (L121+25%PPG) (FIG. 17A) or dual-layer NSC (L121+25%PPG/L121) (FIG. 17B).

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

Illustrative embodiments of the invention are described below. In the interest of clarity, not all features of an actual implementation are described in this specification. It will of course be appreciated that in the development of any such actual embodiment, numerous implementation-specific decisions must be made to achieve the developers' specific goals, such as compliance with system-related and business-related constraints, which will vary from one implementation to another. Moreover, it will be appreciated that such a development effort might be complex and time-consuming, but would be a routine undertaking for those of ordinary skill in the art having the benefit of this disclosure.

DETECTION OF M. TUBERCULOSIS m BIOLOGICAL SAMPLES

Many embodiments described herein relate to the detection of one or more M. tuberculosis-specific biomolecules in a sample. The sample can comprise, or be derived from, at least one biological fluid selected from the group consisting of blood serum, blood plasma, blood, urine, seminal fluid, seminal plasma, pleural fluid, ascites, nipple aspirate, feces, and saliva (see, for example, U.S. Patent Appl. Publ. No. 201 1/0065201, specifically incorporated herein in its entirety by express reference thereto). In particular, the sample may contain, or be derived from, at least one bodily fluid, including, without limitation, sputum, pleural effusion, cerebrospinal fluids, urine, serum and plasma. Preferably the bodily fluid will be obtained from a vertebrate mammal, and in particular, a human having, suspected of having, and/or at risk for developing a bacterial infection, such as that caused by one or more strains or species of Mycobacteria.

Many M. tuberculosis-specific biomolecules are suitable for detection by the methods described herein, including, for example, protein, peptide, polynucleotide and polysaccharide. In some embodiments, the sample may contain at least one ESAT-6- specific protein or peptide, at least one CFP-10-specific protein or peptide, or one or more proteolytic products thereof, each of which has been shown to be a specific biomarker for TB (see e.g., Collins et al, 2005; and Flores et al, 201 1; each of which is specifically incorporated herein in its entirety by express reference thereto).

NANOPOROUS FILMS

Various nanoporous films and fabrication methods therefor suitable for use in the practice of the invention have been recently described in, for example, Bouamrani et al., 2010; Hu et al., 2010; Hu et al., 201 1 ; and U.S. Pat. Appl. Publ. No. 2011/0065207; each of which is incorporated herein in its entirety by express reference thereto).

The nanoporous film can comprise, for example, a plurality of pores, including mesopores having an average diameter of about 2-50 nm. In some embodiments, the nanoporous film comprises a plurality of mesopores having an average diameter of about 2- 20 nm, or about 3-10 nm, or about 6-8 nm. In some embodiments, the nanoporous film comprises pores of substantially the same diameter. In another embodiment, the nanoporous film comprises pores of at least two different diameters.

The porosity of the nanoporous film can be, for example, at least about 40 to about 90%, alternatively, at least about 50 to about 80%, or more preferably still, about 60 to about 70%. The pore morphology can be pre-determined to be, for example, cubic, hexagonal, honeycomb-like, tubular, circular, oblong, and/or combination thereof. In some embodiments, the nanoporous film can comprise at least two domains having pores of substantially different sizes, connectivities, and/or morphologies. In one embodiment, the nanoporous film is positively charged, which facilitates its preference for absorbing negatively-charged biomolecules. In other embodiments, the nanoporous film may be negatively charged, and therefore useful in absorbing to positively- charged biomolecules. In alternative embodiments, the nanoporous film may be fabricated such that it is substantially electrically neutral.

The nanoporous film may contain, for example, one or more nanoporous oxide materials such as a nanoporous silica, a nanoporous titanium oxide, a nanoporous alumina a nanoporous iron oxide, or a nanoporous silicon or a nanoporous carbon, or any combination thereof. The nanoporous film, for example, may be functionalized with one or more organic functional groups, or functionalized with one or more metal ions (see, e.g., U. S. Pat. Appl. Publ. No. 201 1/0065207, specifically incorporated in its entirety by express reference thereto).

The nanoporous film may, for example, be composed of a single-layer nanoporous film, a dual-layer nanoporous film, or even a multi-layer nanoporous film. Dual-layer nanoporous films may include, for example, a first or bottom layer having a first average pore diameter, and a second or top layer having a second average pore diameter that is larger than the first average pore diameter. Such dual- and multi-layer nanoporous films may be fabricated, for example, by serially coating two or more different silicate sol solutions on a single substrate.

In some embodiments, a dual-layer nanoporous film will be fabricated to include a top layer with larger pore size and a bottom layer with smaller pore size to enhance the capillary force of the top layer nanoporous film with the larger pore sizes. One or more full-length antigens may be trapped, for example, in the top layer of the nanoporous film. After washing, a digestion buffer comprising a proteolytic enzyme can be applied to digest the full-length antigens into smaller fragments. At least some of the smaller antigenic digestion fragments can then trapped, for example, in the bottom layer of the nanoporous film, having flowed through to the bottom layer from the top layer.

Optionally, a second wash can be applied to remove the enzyme and certain salts of the digestion buffer. The antigen fragments will remain in the bottom layer, and can then be removed using a suitable elution buffer. In some embodiments, sensitivity of the methods described herein can be improved by using dual- rather than single-layer nanoporous films.

The nanoporous film can be fabricated by, for example, a surfactant-templated sol- gel process. The nanoporous film can be fabricated, for example, on a substrate by one or more deposition methods known to those of ordinary skill in the art. The substrate can be, for example, a silicon wafer, glass wafer, or a metal layer. The nanoporous film can be deposited onto the surface by one or more methods known to those of ordinary skill in the art, including, without limitation, by spin-coating, by dip-coating, or a combination thereof.

In some embodiments, nanoporous film is fabricated from a coating solution comprising at least one silicate sol, at least one tri-block copolymer, and at least one swelling agent, and at least one solvent. The coating solution can be deposited on a silicon wafer by a conventional method, including, for example, spin coating, dip-coating, or the like. The preferential evaporation of the solvent after spin coating or dip-coating drives silica/copolymer self-assembly into a uniform thin film nanophase by increasing the concentration of polymer to exceed the critical micelle concentration. After removing the polymer template by calcination, nanoporous films with narrow nanoscale pore size distribution and high ratio of surface area to pore volume are formed. Optionally, oxygen plasma treatment can be performed to modify the surface of the nanoporous film.

Further, to facilitate the application of samples, at least one gasket can be attached on top of the nanoporous film. The use of gaskets in nanoporous film fabrication is known to those of ordinary skill in the art, and such gaskets can be made of any suitable material, such as, for example, silicone, metal, rubber, fiberglass, polymer, and the like. In some embodiments, a silicone gasket may be used. Such a gasket may contain, for example, a plurality of culture wells. In preferred embodiments, each culture well is fabricated to provide a diameter of about 3-mm, and a height of about 1-mm, although other culture well dimensions are contemplated to fall within the scope of the present disclosure.

ENRICHING M. TUBERCULOSIS-SPECIFIC BIOMOLECULES

In many embodiments described herein, M. tuberculosis-specific biomolecules may be enriched or concentrated using a nanoporous film prior to sample assay and biomarker detection.

In some embodiments, a sample comprising the M. tuberculosis-specific biomolecule is directly applied onto the nanoporous film. The sample can be applied, for example, into a plurality of culture wells formed by a gasket. In other embodiments, a sample comprising the M. tuberculosis-specific biomolecule is indirectly applied onto the nanoporous film through microfluidic channels patterned on the nanoporous film (see, e.g., Hu et ah, 201 1, which is hereby incorporated in its entirety by express reference thereto).

Upon contacting the nanoporous film, the M. tuberculosis -specific biomolecule, due to its size, will be able to enter and reside in the pores of the nanoporous film. The M. tuberculosis-specific biomolecule can be, for example, absorbed to the walls of the pores. The M. tuberculosis-specific biomolecule can absorbed onto the nanoporous film by, for example, van der Waals forces. The M. tuberculosis-specific biomolecule and the pores may be of opposite charge to permit electrostatic interaction between the target and the film itself. The use of oppositely-charged films may increase the overall yield or rate of adsorption, but is not required.

In one embodiment, the nanoporous film may be adapted and configured such that the abundant serum protein, albumin, is substantially excluded from entering the pores. After the sample suspected of containing M. tuberculosis-specific biomolecules is applied to the nanoporous film, the film may be washed one or more times to remove large molecules such as albumin that were not collected and enriched by the nanoporous film. In one embodiment, water may be used for the washing step.

In some embodiments, the sample suspected of comprising one or more M. tuberculosis-specific biomolecules may be "pre-concentrated" before application onto the nanoporous film. M. tuberculosis-specific biomolecules, if present in the sample, can be pre-concentrated by one or more conventional methods, including for example, by precipitating the proteinaceous fraction of the sample. Standard protein precipitation methods are known to those of ordinary in the art and may include, for example, the use of ammonium sulfate. The resulting protein precipitate can then be dissolved in a suitable solvent before being applied onto the nanoporous film.

Proteolysis of M. tuberculosis-Specific Biomolecules

In many embodiments described herein, the sample suspected of containing M. tuberculosis-specific biomolecules may be subjected to proteolysis prior to before detection. The sample may, for example, be digested using one or more proteolytic enzymes, such as a protease or a peptidase, to proteolytically -cleave one or more proteins present in the sample.

In embodiments wherein the biomolecule of interest is an M. tuberculosis '-specific protein or peptide, the sample may be pre-treated using one or more proteases. Various protease or peptidase are known in the art, including, for example, serine proteases, cysteine proteases, aspartate proteases, threonine proteases, glutamic acid proteases, and metalloproteases

(Rawlings, et ah, 2010). In particular embodiments, the protease is preferably trypsin or an analog or active fragment thereof.

In some embodiments, the M. tuberculosis-specific biomolecule can be digested directly on or within the nanoporous film itself (i.e., "on-chip digestion" or on-site proteolysis). The proteolytic products so produced can then be extracted from the nanoporous film for further characterization or analysis. Extraction of the digestion products may be achieved by, for example, using one or more suitable elution buffers. In other embodiments, the biomolecules of interest, or the proteolytic byproducts thereof may be extracted from the nanoporous film before being digested at a different site.

The digestion product can comprise, for example, at least one, at least two, or least three different identifiable fragments of a M. tuberculosis-specific biomolecule. In some embodiments where the M. tuberculosis-specific biomolecule is a protein or peptide, the digestion product can comprise, for example, at least one, at least two, or least three different identifiable peptides resulting from proteolytic cleavage of the molecule.

In some embodiments where the M. tuberculosis-specific biomolecule is an ESAT-6 protein or peptide, the digestion product can comprise, for example, at least one peptide having a mass fingerprint at about 1895-1910 Da. The digestion product(s) can include, for example, at least one ESAT-6 fragment comprising, consisting essentially of, or alternatively, consisting of, the sequence of WDATATEL ALQNLAR (SEQ ID NO: l), at least one ESAT-6 fragment comprising, consisting essentially of, or alternatively, consisting of, the sequence of LAAAWGGSGSEAYQGVQQK (SEQ ID O:2), or a combination of both fragments.

In some embodiments, wherein the M. tuberculosis-specific biomolecule is a CFP- 10 protein or peptide, the digestion product can include, for example, at least one CFP-10 fragment resulting from proteolysis. In such embodiments, the digestion product(s) can include, for example, at least one CFP-10-specific peptide fragment that comprises, consists essentially of, or alternatively, consists of, the amino acid sequence of any one of: TQIDQVESTAGSLQGQWR (SEQ ID O:3), ADEEQQQALSSQMGF (SEQ ID O:4), TDAATLAQEAGNFER (SEQ ID O:5), GAAGTAAQAAVVR (SEQ ID O:6) and QAGVQYSR (SEQ ID NO:7). In certain embodiments, the digestion products can include, for example, at least two CFP-10-specific peptide fragments, each of which can comprise, consist essentially of, or alternatively, consist of, an amino acid sequence as set forth in any one of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6 and SEQ ID NO:7.

Likewise, in other embodiments, the digestion products can include, for example, at least three, at least four, or at least CFP-10-specific peptide fragments, each of which comprising, consisting essentially of, or alternatively, consisting of, an amino acid sequence as set forth in any one of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6 and SEQ ID NO: 7. Detecting M. tuberculosis-Specific Biomolecules

In many embodiments described herein, the presence of a M. tuberculosis-specific biomolecule can be detected by any one or more techniques known to those of ordinary skill in the art, including, without limitation, mass spectrometry, gel electrophoresis, chromatography, one or more bioassays, one or more immunological assays, or a combination of two or more such techniques. In the practice of the invention, mass spectrometry has been preferably used to detect the presence of M. tuberculosis-specific proteins and peptides from a sample of interest. Exemplary mass spectrometry methods include, without limitation, matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry, liquid chromatography-mass spectrometry (LC-MS) mass spectrometry, ESI-MS mass spectrometry, tandem mass spectrometry, and/or surface- enhanced laser desorption/ionization (SELDI) mass spectrometry. In one embodiment, MALDI-TOF MS may be employed to readily detect the presence of one or more M. tuberculosis-specific biomolecules. In another embodiment, SELDI is used to detect the presence of the M. tuberculosis-specific biomolecule. In a further embodiment, LC-MS is used to detect the presence of a tuberculosis-specific biomolecule.

The presence of a M. tuberculosis-specific biomolecule can be detected by, for example, finding at least one, at least two, or at least three or mass spectroscopy "fingerprints" unique to the particular biomolecule of interest. The presence of one or more M. tuberculosis-specific biomolecules in a sample can be detected by, for example, finding at least one, at least two, or at least three or more mass fingerprints of one or more enzymatic digestion products of the particular biomolecule of interest.

In some embodiments, wherein the M. tuberculosis-specific biomolecule is an ESAT-6-specific protein or peptide, the presence of ESAT-6 can be detected by, for example, finding at least one mass fingerprint of an ESAT-6 protein or one or more of its proteolytic fragments. The presence of an ESAT-6-specific molecule can be detected by, for example, identifying a mass fingerprint at about 1895-1910 Da, or more precisely, a mass fingerprint at about 1900.951 1 Da ([M+H] ⁺ ). Similarly, the presence of an ESAT-6- specific molecule can be detected by, for example, identifying a mass fingerprint at about 1907.9246 Da ([M+H] ⁺ ).

Alternatively, where the M. tuberculosis-specific biomolecule is a CFP-10-specific protein or peptide, the presence of the CFP-10 biomarker can be detected by, for example, identifying at least one mass fingerprint of a CFP-10 protein or one or more of its proteolytic fragments. The presence of CFP-10 can be detected by, for example, identifying a mass fingerprint at about 2003.9781 Da ([M+H] ⁺ ), at about 1668.7170 Da ([M+H] ⁺ ), or at about 1593.7503 Da ([M+H] ⁺ ). The presence of a CFP-10-specific protein or peptide can also be confirmed by identifying, for example, a mass fingerprint at about 1142.6276 Da ([M+H] ⁺ ), or at about 908.4584 Da ([M+H] ⁺ ).

In one embodiment, at least one M. tuberculosis-specific biomolecule is detected to identify active TB, such as ESAT-6 or CFP-10. In other embodiments, at least two M. tuberculosis-specific biomolecules may be detected within a sample to identify the presence of TB organisms, such as ESAT-6 and CFP-10. In further embodiments, three or more M. tuberculosis-specific biomolecules may be detected to confirm the presence of TB in a sample.

EXEMPLARY METHODS

FIG. 1A illustrates an exemplary detection procedure for ESAT-6 and/or CFP-10. The biological samples can be applied to a gasket culture well attached on top of a nanoporous silica film. The nanoporous silica films can be fixed on a flat substrate. The fractionation process can be completed after serial washes. The relatively small size of ESAT-6 (molecular weight: 10 kDa) allows it to be captured by the silica nanopores that have an average diameter in the range of about 5, about 6, about 7 or about 8 or so nm.

To identify ESAT-6 and/or CFP-10 from biological fluids with MALDI TOF MS, mass fingerprinting may be performed. Because smaller proteins or peptide species provide higher signals and resolution in mass spectrometry, a proteolytic enzyme, such as trypsin, may be used to pre-treat the sample, and to cleave full-length ESAT-6 and/or CFP-10 proteins into smaller peptide sub-fragments, which can then be detected by suitable methods.

One advantage of the instant method is that unlike conventional digestion processes, which are usually conducted in bulk solution, the proteolytic enzyme(s) can be applied directly onto the nanoporous silica film, where they can interact with the sample inside the nanopores. This on-chip nanobiocatalysis process provides many advantages over solution- based proteolysis, including higher efficiency and better stability. In addition, the on-chip digestion protocol can eliminate several additional steps, including protein extraction from the silica nanopore and buffer exchange for enzymatic digestion, and thus further simplify the overall diagnostic assay. ADDITIONAL APPLICATIONS

While the present invention has been optimized to detect biomolecules that are specific for MT and the detection of TB-causing organisms, the methods and apparatus described herein can also be used to detect other biomolecules of interest.

In certain applications, the target protein is specific to one or more pathogens associated with a particular infectious disease. In a manner analogous to that demonstrated herein for ESAT-6 and CFP-10 proteins, the particular pathogen-specific protein of interest may be digested with one or more proteases to produce one or more peptide fragments, at least one of which comprises a unique mass spectral fingerprint, and those fingerprints can be detected via mass spectrometry, such as MALDI-TOF MS.

The methods and kits described herein possess broad applicability in the molecular arts, since many infectious diseases have been linked to specific microorganisms, many of which have known type- or species-specific biomarkers, which may be detected in a manner analogous to that demonstrated herein for TB-specific biomarkers. Exemplary pathogens suitable for detection using the disclosed methods include, but are not limited to, bacterial pathogens, viral pathogens, fungal pathogens, unicellular eukaryotic pathogens such as protozoans, spirochetes, prions, or other pathogenic microbiological organisms. Specific examples include, without limitation, the detection of viral pathogens such as HSV, HIV, West Nile Virus, hantavirus, Hepatitis A, Hepatitis B, Norovirus, poliovirus, Rotavirus, etc, the detection of bacterial pathogens such as the causal agents of pneumonia, Legionnaire's disease, food poisoning, food infection, food intoxication, diphtheria, Lyme disease, and/or the detection of protozoal pathogens, including, without limitation, those of the genus Plasmodium. DIAGNOSTIC KITS

Kits including one or more of the disclosed pathogen-specific biomarkers or pharmaceutical formulations including such; and instructions for using the kit in a diagnostic, therapeutic, prophylactic, and/or other clinical embodiment(s) also represent preferred aspects of the present disclosure. Such kits may include one or more of the disclosed pathogen-specific biomarkers, either alone, or in combination with one or more additional diagnostic compounds, pharmaceuticals, and such like. The kits according to the invention may be packaged for commercial distribution, and may further optionally include one or more delivery, storage, or assay components. The container(s) for such kits may typically include at least one vial, test tube, flask, bottle, syringe or other container, into which the pathogen-specific biomarker composition(s) may be placed. Alternatively, a plurality of distinct biomarker composition(s) and/or distinct proteolytic enzymes may be prepared in a single formulation, and may be packaged in a single container, vial, flask, syringe, catheter, cannula, bottle, test tube, ampoule, or other suitable container. The kit may also include a larger container, such as a case, that includes the containers noted above, along with other equipment, instructions, and the like.

For example, a kit can be provided comprising two or more of the following:

(i) a nanoporous film disposed on a solid substrate adapted for accepting a human body fluid sample and enriching at least one target protein therefrom, wherein the nanoporous film comprises a plurality of pores in which the enriched target protein resides, (ii) a digestion buffer comprising at least one protease adapted for digesting the target protein to produce at least one fragment of the target protein having a mass fingerprint detectable in mass spectrometry,

(iii) elution buffer adapted for extracting the at least one fragment of the target protein from the nanoporous film,

(iv) a washing buffer adapted for washing the nanoporous film before the digestion buffer is added onto the nanoporous film, (v) instructions to use the kit.

The nanoporous film can be, for example, a silica film comprising a plurality of pores having an average diameter of about 3 to about 10 nm, and more preferably, a silica film comprising a plurality of pores having an average diameter of about 6 to about 8 nm, wherein the digestion buffer comprises a first proteolytic enzyme, such as trypsin, and wherein the kit comprises at least one gasket attached onto the nanoporous film to form a plurality of wells.

One embodiment, for example, provides a kit, comprising (i) a nanoporous film disposed on a solid substrate adapted for accepting a sample of a human bodily fluid, and enriching at least one target biomarker protein or peptide therefrom, wherein the nanoporous film comprises a plurality of pores in which the enriched target protein resides, and (ii) a digestion buffer comprising at least one protease adapted for digesting the target protein to produce at least one fragment of the target protein having a mass fingerprint detectable in mass spectrometry. In one embodiment, the kit can further comprise an elution buffer adapted for extracting the at least one fragment of the target protein from the nanoporous film. In one embodiment, the kit can further comprise a washing buffer adapted for washing the nanoporous film before the digestion buffer is added onto the nanoporous film.

The kit can be part of a larger system. For example, the system can also include an instrument such as, for example, a mass spectrometry device for detecting said mass fingerprint. Sample preparation items can also be included in the various systems and kits.

EXEMPLARY DEFINITIONS

The terms "about" and "approximately" as used herein, are interchangeable, and should generally be understood to refer to a range of numbers around a given number, as well as to all numbers in a recited range of numbers (e.g., "about 5 to 15" means "about 5 to about 15" unless otherwise stated). Moreover, all numerical ranges herein should be understood to include each whole integer within the range.

As used herein, the term "carrier" is intended to include any solvent(s), dispersion medium, coating(s), diluent(s), buffer(s), isotonic agent(s), solution(s), suspension(s), colloid(s), inert(s) or such like, or a combination thereof that is pharmaceutically acceptable for administration to the relevant animal or acceptable for a therapeutic or diagnostic purpose, as applicable.

As used herein, the terms "protein," "polypeptide," and "peptide" are used interchangeably, and include molecules that include at least one amide bond linking two or more amino acid residues together. Although used interchangeably, in general, a peptide is a relatively short (e.g., from 2 to about 100 amino acid residues in length) molecule, while a protein or a polypeptide is a relatively longer polymer (e.g., 100 or more residues in length). However, unless specifically defined by a chain length, the terms peptide, polypeptide, and protein are used interchangeably.

As used herein, an "antigenic polypeptide" or an "immunogenic polypeptide" is a polypeptide which, when introduced into a vertebrate, reacts with the vertebrate's immune system molecules, i.e., is antigenic, and/or induces an immune response in the vertebrate, i.e., is immunogenic. As used herein, the term "buffer" includes one or more compositions, or aqueous solutions thereof, that resist fluctuation in the pH when an acid or an alkali is added to the solution or composition that includes the buffer. This resistance to pH change is due to the buffering properties of such solutions, and may be a function of one or more specific compounds included in the composition. Thus, solutions or other compositions exhibiting buffering activity are referred to as buffers or buffer solutions. Buffers generally do not have an unlimited ability to maintain the pH of a solution or composition; rather, they are typically able to maintain the pH within certain ranges, for example from a pH of about 5 to 7.

As used herein, the term "epitope" refers to that portion of a given immunogenic substance that is the target of, i.e., is bound by, an antibody or cell-surface receptor of a host immune system that has mounted an immune response to the given immunogenic substance as determined by any method known in the art. Further, an epitope may be defined as a portion of an immunogenic substance that elicits an antibody response or induces a T-cell response in an animal, as determined by any method available in the art (see, for example, Geysen et ah, 1984). An epitope can be a portion of any immunogenic substance, such as a protein, polynucleotide, polysaccharide, an organic or inorganic chemical, or any combination thereof. The term "epitope" may also be used interchangeably with "antigenic determinant" or "antigenic determinant site."

As used herein, the term "patient" (also interchangeably referred to as "host" or

"subject") refers to any host that can serve as a recipient of one or more of the therapeutic or diagnostic formulations as discussed herein. In certain aspects, the patient is a vertebrate animal, which is intended to denote any animal species (and preferably, a mammalian species such as a human being). In certain embodiments, a "patient" refers to any animal host, including but not limited to, human and non-human primates, avians, reptiles, amphibians, bovines, canines, caprines, cavines, corvines, epines, equines, felines, hircines, lapines, leporines, lupines, murines, ovines, porcines, racines, vulpines, and the like, including, without limitation, domesticated livestock, herding or migratory animals or birds, exotics or zoological specimens, as well as companion animals, pets, and any animal under the care of a veterinary practitioner.

The term "e.g.," as used herein, is used merely by way of example, without limitation intended, and should not be construed as referring only those items explicitly enumerated in the specification. As used herein, the term "polypeptide" is intended to encompass a singular "polypeptide" as well as plural "polypeptides," and includes any chain or chains of two or more amino acids. Thus, as used herein, terms including, but not limited to "peptide," "dipeptide," "tripeptide," "protein," "enzyme," "amino acid chain," and "contiguous amino acid sequence" are all encompassed within the definition of a "polypeptide," and the term "polypeptide" can be used instead of, or interchangeably with, any of these terms. The term further includes polypeptides that have undergone one or more post-translational modification(s), including for example, but not limited to, glycosylation, acetylation, phosphorylation, amidation, derivatization, proteolytic cleavage, post-translation processing, or modification by inclusion of one or more non-naturally occurring amino acids. Conventional nomenclature exists in the art for polynucleotide and polypeptide structures. For example, one-letter and three-letter abbreviations are widely employed to describe amino acids: Alanine (A; Ala), Arginine (R; Arg), Asparagine (N; Asn), Aspartic Acid (D; Asp), Cysteine (C; Cys), Glutamine (Q; Gin), Glutamic Acid (E; Glu), Glycine (G; Gly), Histidine (H; His), Isoleucine (I; He), Leucine (L; Leu), Methionine (M; Met), Phenylalanine (F; Phe), Proline (P; Pro), Serine (S; Ser), Threonine (T; Thr), Tryptophan (W; Trp), Tyrosine (Y; Tyr), Valine (V; Val), and Lysine (K; Lys). Amino acid residues described herein are preferred to be in the "1" isomeric form. However, residues in the "d" isomeric form may be substituted for any 1-amino acid residue provided the desired properties of the polypeptide are retained.

"Protein" is used herein interchangeably with "peptide" and "polypeptide," and includes both peptides and polypeptides produced synthetically, recombinantly, or in vitro and peptides and polypeptides expressed in vivo after nucleic acid sequences are administered into a host animal or human subject. The term "polypeptide" is preferably intended to refer to all amino acid chain lengths, including those of short peptides of from about 2 to about 20 amino acid residues in length, oligopeptides of from about 10 to about 100 amino acid residues in length, and polypeptides including about 100 amino acid residues or more in length. The term "sequence," when referring to amino acids, relates to all or a portion of the linear N-terminal to C-terminal order of amino acids within a given amino acid chain, e.g., polypeptide or protein; "subsequence" means any consecutive stretch of amino acids within a sequence, e.g., at least 3 consecutive amino acids within a given protein or polypeptide sequence. With reference to nucleotide and polynucleotide chains, "sequence" and "subsequence" have similar meanings relating to the 5' to 3' order of nucleotides. As used herein, "an effective amount" would be understood by those of ordinary skill in the art to provide a therapeutic, prophylactic, or otherwise beneficial effect against the organism, its infection, or the symptoms of the organism or its infection, or any combination thereof.

The phrases "isolated" or "biologically pure" refer to material that is substantially, or essentially, free from components that normally accompany the material as it is found in its native state. Thus, isolated polynucleotides in accordance with the invention preferably do not contain materials normally associated with those polynucleotides in their natural, or in situ, environment.

As used herein, the term "kit" may be used to describe variations of the portable, self-contained enclosure that includes at least one set of reagents, components, or pharmaceutically-formulated compositions to conduct one or more of the diagnostic or therapeutic methods of the invention.

The term "sequence," when referring to amino acids, relates to all or a portion of the linear N-terminal to C-terminal order of amino acids within a given amino acid chain, e.g., polypeptide or protein; "subsequence" means any consecutive stretch of amino acids within a sequence, e.g., at least 3 consecutive amino acids within a given protein or polypeptide sequence. With reference to nucleotide chains, "sequence" and "subsequence" have similar meanings relating to the 5' to 3' order of nucleotides.

As used herein, "heterologous" is defined in relation to a predetermined referenced

DNA or amino acid sequence. For example, with respect to a structural gene sequence, a heterologous promoter is defined as a promoter that does not naturally occur adjacent to the referenced structural gene, but which is positioned by laboratory manipulation. Likewise, a heterologous gene or nucleic acid segment is defined as a gene or segment that does not naturally occur adjacent to the referenced promoter and/or enhancer elements.

As used herein, the term "homology" refers to a degree of complementarity between two polynucleotide or polypeptide sequences. The word "identity" may substitute for the word "homology" when a first nucleic acid or amino acid sequence has the exact same primary sequence as a second nucleic acid or amino acid sequence. Sequence homology and sequence identity can be determined by analyzing two or more sequences using algorithms and computer programs known in the art. Such methods may be used to assess whether a given sequence is identical or homologous to another selected sequence.

As used herein, "homologous" means, when referring to polypeptides or polynucleotides, sequences that have the same essential structure, despite arising from different origins. Typically, homologous proteins are derived from closely related genetic sequences, or genes. By contrast, an "analogous" polypeptide is one that shares the same function with a polypeptide from a different species or organism, but has a significantly different form to accomplish that function. Analogous proteins typically derive from genes that are not closely related.

As used herein, the term "substantially homologous" encompasses sequences that are similar to the identified sequences such that antibodies raised against peptides having the identified sequences will specifically bind to peptides possessing the "substantially homologous" amino acid sequence. In some variations, the amount of detectable antibodies induced by the homologous sequence is identical to the amount of detectable antibodies induced by the identified sequence. In other variations, the amounts of detectable antibodies induced are substantially similar, thereby providing immunogenic properties. For example, "substantially homologous" can refer to at least about 75%, preferably at least about 80%, and more preferably at least about 85% or at least about 90% identity, and even more preferably at least about 95%, more preferably at least about 97% identical, more preferably at least about 98% identical, more preferably at least about 99% identical, and even more preferably still, at least substantially or entirely 100% identical (i.e., "invariant").

The terms "identical" or percent "identity," in the context of two or more peptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues that are the same, when compared and aligned for maximum correspondence over a comparison window, as measured using a sequence comparison algorithm or by manual alignment and visual inspection.

The phrases "isolated" or "biologically pure" refer to material that is substantially, or essentially, free from components that normally accompany the material as it is found in its native state. Thus, isolated peptides in accordance with the invention preferably do not contain materials normally associated with the peptides in their in situ environment.

As used herein, "mammal" refers to the class of warm-blooded vertebrate animals that have, in the female, milk-secreting organs for feeding the young. Mammals include without limitation humans, apes, many four-legged animals, whales, dolphins, and bats. A human is a preferred mammal for purposes of the invention.

"Link" or "join" refers to any method known in the art for functionally connecting peptides, including, without limitation, recombinant fusion, covalent bonding, disulfide bonding, ionic bonding, hydrogen bonding, electrostatic bonding, and such like. The term "pathogen" is defined herein as any sort of infectious agent, including e.g., viruses, prions, protozoans, parasites, as well as microbes such as bacteria, yeast, molds, fungi, and the like.

The term "about," as used herein, should generally be understood to mean "approximately", and typically refers to numbers approximately equal to a given number recited within a range of numerals. Moreover, all numerical ranges herein should be understood to include each whole integer within the range.

In accordance with long standing patent law convention, the words "a" and "an" when used in this application, including the claims, denotes "one or more."

EXAMPLES

The following examples are included to demonstrate illustrative embodiments of the invention. It should be appreciated by those of ordinary skill in the art that the techniques disclosed in these examples represent techniques discovered to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of ordinary skill in the art should, in light of the present disclosure appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

EXAMPLE 1 -ESAT-6 DETECTION

Fabrication and Characterization of Nanoporous Silica Thin Film

The fabrication of nanoporous silica thin film is described in Bouamrani et al., 2010; Hu et al, 2010; Hu et al, 201 1; and U.S. Patent Appl. Publ. No. 201 1/0065207. Briefly, the coating silicate sol was prepared by adding 14 mL of tetraethyl orthosilicate (TEOS) into a mixture of 17 mL of ethanol, 6.5 mL of distilled water, and 0.5 mL of 6 M HC1 and stirred for 2 hr at 80°C to form a clear silicate sol. After cooling to room temperature, 10 mL of silicate sol was added to a mixture of 1.2 g of Pluronic L121, 10 mL of ethanol, and differing amounts of polypropylene glycol (PPG). The coating solution was stirred at room temperature for 2 hr, and deposited on a Si(100) wafer by spin-coating at a spin rate of 2500 rpm for 20 sec. To increase the degree of polymerization of the silica framework in the films, and to further improve their thermal stability, the as-deposited films were heated at 80°C for 12 hr. The films were then calcinated at 450°C for 5 hr to remove the organic compound. The temperature was raised at l°C/min. Pluronic L121 was obtained from BASF. All other chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA).

The thickness and porosity of nanoporous silica films were characterized by variable angle spectroscopic ellipsometry (J. A. Woollam Co. M-2000DI). The thickness of the thin films and their porosities were calculated using both Cauchy and effective medium approximation (EMA) models with CompleteEASE software (version 4.58). Ellipsometric optical quantities were detected by acquiring spectra at 55, 60, and 65° incidence angles, in a wavelength ranging from 300 to 1800 nm. All fabricated porous silica thin films were characterized by scanning over the entire 4-inch wafer by ellipsometry. The variation of porosity and thickness was less than 0.5%.

On-Chip Fractionation and Digestion ofESAT-6

Normal human serum was obtained from Valley Biomedical (Winchester, VA, USA). Recombinant ESAT-6 was purchased from Diagnostics, Inc. (Woburn, MA, USA). As shown in FIG. 1A, FIG. IB, and FIG. 1C, 6 of protein solutions was pipetted onto the silica nanoporous film and incubated for 30 min in a humidified chamber at room temperature. The protein solution was then discarded, and 10 of deionized water was applied onto the silica porous film to wash away larger proteins. The washing process was then repeated 3 times. For enzymatic digestion, 10 of 0.01 mg/mL trypsin dissolved in 100 mM sodium bicarbonate was applied onto the silica nanoporous film and incubated overnight at 37°C. 10 of elution buffer (0.1% trifluoroacetic acid [TFA] + 50% acetonitrile [ACN]) was then pipetted to extract the protein fragments. The elution buffer was then removed and stored in microcentrifuge tubes for MALDI-TOF analysis. To test the sensitivity of ESAT-6 detection, low concentration of ESAT-6 solution were concentrated by precipitation using ammonium sulfate. The concentrated protein pellet was dissolve in 20 μΐ _^ of 100 mM sodium bicarbonate. To help dissolve protein, 16 M urea was added into the solution to reach 2 M final concentration.

MALDI-TOF Analysis of ESAT-6

Matrix-assisted laser desorption/ionization time-of flight mass spectrometry

(MALDI TOF MS) has been described in Petricoin et al, J Natl Cancer I 2002, 94 (20), 1576-1578; Petricoin et al, Lancet 2002, 359 (9306), 572-577; Petricoin et al, Curr Opin Biotech 2004, 15 (1), 24-30; and Hortin, Clin Chem 2006, 52 (7), 1223-1237, all of which are incorporated by reference in their entireties. Here, a matrix solution of 4 g/L of a-cyano-4-hydroxycinnamic acid (HCCA) was prepared in the solution of ACN and water (1 : 1, vol/vol.) which contained 0.1% TFA. 0.5 μϊ _^ of each sample was spotted onto the MALDI target plate first, waiting to dry at room temperature. Next, 0.5 μϊ _^ of the matrix solution was spotted onto the target plate and allowed to dry at room temperature. Mass spectra were then collected using a MALDI TOF/TOF Analyzer (Model 4700, Applied Biosystems), in the positive reflectron mode in the range of 800-5000 Da. Mass spectra were acquired from 5000 laser shots under 4300 laser intensity, and calibrated externally using a peptide calibration standard. Raw spectra were processed with Data Explorer software (Applied Biosystems).

XPS (X-Ray Photoelectron Spectroscopy) Depth Profiling

10 μΜ of ESAT-6 dissolved in 100 mM NaCl was incubated on 6-nm and 8-nm NSCs, and then washed with deionized water. NSCs were incubated in a vacuum chamber overnight prior to XPS measurement. PHI Quantera XPS equipped with Ar+ ion gun was used to construct concentration depth profiles. The Ar+ ion sputtered NSCs at accelerating voltage 3 kV in a 2 x 2 mm area. Because the thickness of silica film was determined by ellipsometry, the etching rate on porous silica could be calibrated by sputtering until oxygen (Ols) signal vanished. A 9-sec sputtering time interval was employed to reach a 5.25-nm depth spacing with a 35 nm/min Ar+ ion etching rate. Nitrogen (Nls) spectra were observed to identify the amount of ESAT-6 trapped at different depths (see FIG. 6A, FIG. 6B, and FIG. 6C).

Pre-concentration of ESAT-6

1 mL of each concentration of ESAT-6 in urine was mixed with 0.4 g of ammonium sulphate. The solution was vortexed for 30 min, the precipitated proteins were collected by benchtop centrifugation, and the supernatant was then removed. 20 μϊ _^ of 100 mM ammonium bicarbonate and 2 μΕ of 16 M urea were used to dissolve the precipitated proteins. The concentrated protein solution was then directly applied to NSCs for on-chip fractionation.

Indirect ELISA of ESAT-6 - Comparative Example

Recombinant ESAT-6, mouse monoclonal antibody against ESAT-6, and indirect ELISA kits were all obtained from Abeam, Inc. To test the sensitivity of indirect ELISA of ESAT-6 in different human bodily fluids, a series of concentration of recombinant ESAT-6 dissolved in IX PBS buffer, urine, and 5% human serum in IX PBS buffer was prepared. The manufacturer's standard protocol for indirect ELISA was followed: the antigens were first coated on microplates by incubation at 4°C overnight. The remaining protein-binding sites were blocked by 5% serum in IX PBS buffer. The antigen was incubated with primary antibody, and then conjugated with secondary antibody. 3,3',5,5'-tetramethylbenzidine (TMB) was used as a detection reagent.

EXAMPLE 2 -ESAT-6/CFP-10 DETECTION ANALYSES

Detection of ESA T-6

An exemplary detection procedure in accordance with one aspect of the present invention is illustrated in FIG. 1A. The biological samples were applied to a silicone gasket culture well (3 -mm diameter and 1-mm height) attached on top of the nanoporous silica film. The fractionation process was completed after serial washes as described in Example 1.2. Because the silica films were fixed on the flat substrate, the fractionation process was easily applied without any inconvenient washing procedure, such as sedimentation steps usually required in particle-based systems. The relatively small size of ESAT-6 (molecular weight: lO kDa) allowed it to be captured by the silica nanopores. To identify ESAT-6 protein from biological fluids with MALDI TOF MS, a mass fingerprinting must first be established. Because smaller protein or peptides species provide higher signals and resolution in mass spectrometry and full length ESAT-6 (lO kDa) is relatively large for MALDI TOF MS under linear mode (FIG. 4), the proteolytic enzyme, trypsin, was used to cleave full length ESAT-6 into smaller fragments. In contrast to conventional digestion processes that are usually conducted in bulk solution, the digestive enzymes in this case were applied directly onto the nanoporous silica film and they interacted with protein inside the nanopores. Several advantages of this novel nanobiocatalysis process over conventional solution-based digestion have been reported, including higher efficiency and better stability (Kim et al, 2010; Qiao et al, 2008; and Savino et al, 2011). In addition, the on-chip digestion protocol eliminated several additional processes, including protein extraction from the silica nanopore and buffer exchange for enzymatic digestion can further simplify the operation procedures.

After 10 hours of incubation, the protein retained inside the silica pores was trypsin digested into smaller fragments that were then extracted with the elution buffer. The fingerprinting spectrum of ESAT-6 showed strong signal from two major fragments corresponding to mass [M+H] ⁺ 1900.951 1 Da and 1907.9246 Da. Liquid chromatography- mass spectrometry was used to confirm that these two fragments did indeed originate from proteolysis of ESAT-6 protein (1900.951 1 m/z, amino acid sequence: WDATATEL ALQNLAR [SEQ ID O: l]; 1907.9246 m/z, amino acid sequence: LAAAWGGSGSEAYQGVQQK, [SEQ ID NO:2]). Although these fragments did not represent the only degradation products from trypsin digestion of ESAT-6, these two peptides presented the strongest mass peaks due to their specific physiochemical properties. The results suggested that these two fragments were excellent markers for ESAT-6.

To examine the fractionation capability of the nanoporous silica film, the recombinant ESAT-6 was mixed with human serum and then treated with or without on- chip separation, followed by digestion with trypsin, as shown in FIG. 2A, FIG. 2B, FIG. 2C, and FIG. 2D. Without fractionation, the signal of the major ESAT-6 fragment, 1900.951 1 [M+H] ⁺ at a high concentration of 80 μΜ of ESAT-6, was obscured (FIG. 2A). With on-chip fractionation, the detection of ESAT-6 fragment from the same complex biological fluid was significantly improved (FIG. 2B). Human serum without ESAT-6 was also fractionated and digested on nanoporous silica film as a control. No fragments were identified in the range from 1895 to 1910 Da (FIG. 5). These results indicate that the most abundant species in human serum that may overlap with the ESAT-6 fragments were removed by the fractionation process, which enabled the enrichment and quantification of ESAT-6 from human serum using mass spectrometry.

Optimization of Pore Size

Concentration gradient is a major driving force of protein diffusion into nanopores. Interactions between proteins and the silica surface, especially electrostatic interactions, can play a significant role in protein absorption, and can impact the isolation process (Deere et al, 2002; Katiyar et al, 2005). In addition, the architecture of the nanoporous silica films and the shape and size of target proteins can influence the capturing effects. Previous studies have indicated that the effect of pore blockage by adsorbed proteins is eliminated when the pore diameter is twice as large as the hydrodynamic size of protein (Vinu et al, 2004). Large pores, however, may also enrich large and abundant peptides that can reduce the sensitivity of target LMW molecule in MALDI TOF MS. Furthermore, during the on- chip digestion process, trypsin interacts with either captured protein within the pore, given sufficient space for proper operation, or proteins that have been released back into solution, indicating that pore size will also influence on-chip digestion efficiency. In this context, various pore sizes were tested for ESAT-6 enrichment by adding a swelling agent, polypropylene glycol (PPG), which interacts with the hydrophobic domain to expand the micelle template during nanoporous silica film fabrications (Hu et ah, 2010; Sorensen et ah, 2008). Pluronic triblock copolymer L121 mixed with PPG at 0, 25, 50 and 100 wt% resulted in approximately 5-, 6-, 7- and 8-nm pore diameters, respectively. After performing the fractionation process and on-chip digestion using the nanoporous silica films with different pore size, the highest intensity of 1900.9511 [M+H] ⁺ Da fragment was observed in the 6-nm pores (FIG. 2C). Despite the larger pore size having less steric obstructions for protein diffusion, the higher enrichment efficiency of a 6-nm pore can be attributed to the higher number of pores per unit surface area.

The porosity of nanoporous silica film was measured by ellipsometry as described in Example 1. The normalized number of pores per unit surface area was evaluated by porosity, as reported in Table 1. There were 1.6 times as many 6-nm pores per unit surface area in the nanoporous silica thin-films than there were in films with 8-nm pores. After normalizing of the 1900.951 1 [M+H] ⁺ fragment peaks to the number of pores per unit surface area, no major distinction was observed between 6, 7 and 8 nm pore sizes (FIG. 2D). The 5-nm pores, however, showed only one-fifth the intensity of 6-nm pores. This may attributable to the geometry of ESAT-6 that exhibits a rod-like structure with dimensions of 1.5 x 6 nm (see Renshaw et ah, 2005). The long backbone (rod-like) of the ESAT-6 molecule produces anisotropic diffusion that may reduce diffusion rates inside silica nanopores; especially in the smaller 5 nm pores, where the ESAT-6 and the pores are similar in size (Eichmann et ah, 201 1). In addition, 5-nm pores are also similar in size to trypsin, a globular-like protein with a 4.5 nm diameter (Leiros et ah, 2004). The low diffusion rate of trypsin in these smaller pores would reduce the digestion efficiency inside the nanopore.

TABLE 1

PROPERTIES OF NANOPOROUS SILICA CHIPS pore size porosity pore cross section normalized number of

(nm) (%) area (nm ² ) pores per area*

L121 5 52 19.63 1

L121+25% PPG 6 61 28.27 0.81

L 121+50% PPG 7 64 38.485 0.63

L121+100% PPG 8 68 50.27 0.51

*Number of pores per area calculated based on porosity and pore size. Adapting Nanopore Morphology In fluences CFP-10 Enrichment

Different design parameters (e.g., pore size and shape, chemistry, porosity, etc.) dictate the "landscape" and ultimately the peptidic fingerprint of samples processed by on- chip fractionation. To determine the optimal morphology for CFP-10 isolation, we adjusted the pore morphology by using different copolymers and the swelling agent, polypropylene glycol (PPG), which interacts with the hydrophobic domain of polymers to expand the micelle template during NPS film fabrications. Mixing various compositions of the pluronic triblock copolymers F127, L64, and L121mixed with PPG at 0, 25, 50 and 100 % weight of the copolymers yielded a number of film thickness, porosity, surface area, pore volume and sizes (FIG. 3A).

The isolation efficiency of recombinant CFP-10 was first analyzed as a function of

NPS configurations. 0.05 mg/mL of CFP-10 dissolved in PBS buffer was incubated in the silicon gasket well (3 -mm diameter) pasted on NPS surface. After extensive washes, the amount of CFP-10 remaining in the wash solution was quantified using indirect ELISA, and the percentage of CFP-10 retained in the morphologically distinct nanopores was calculated (FIG. 3B). Significantly lower isolation efficiency was observed when the highly-ordered nanoporous film (F127) was used for fractionation, compared to the use of non-ordered nanoporous films (L121), although the average nanopore sizes of both films were comparable (pore size of F127: 3.7 nm vs. L121 : 3.9 nm). It was previously shown that the F127 film consists of 2-dimensional (2-D) hexagonal and closely -packed nanopores that are perpendicular to the film's surface. In contrast, L121 or L121+PPG films are composed of non-ordered, or worm-like, nanoporous structures. These results suggested that a non- ordered nanopore structure was more conducive to isolating CFP-10. Pore size also strongly influences the fractionation efficiency. Among the non- ordered nanoporous films, L64 film with 3.2-nm average nanopore diameter displays lower isolation efficiency than other films (L121 & L121+PPG). Moreover, the films consisting of nanopore size above 3.9 nm showed similar CFP-10 isolation efficiencies that were irrespective of the addition of PPG (L121 and L121+PPG). This result suggested that the rod-like CFP-10 with dimensions of 1.5 nm x 6 nm was not significantly excluded by nanopores larger than 3.9 nm.

Modifying the nanopore film thickness, without interfering with pore morphology, also influences the efficiency of sample peptide retention and enrichment. The thickness can be manipulated by diluting the coating sol, which is the silicate sol mixed with polymer in ethanol, or by controlling the rotational speed of the spin coater. L121+25% PPG films were synthesized in two varieties: a 643 -nm ("thick") version and a 196-nm ("thin") version. It was observed that the "thick" film captured more CFP-10 peptides. To better understand this phenomenon, the amount of CFP-10 penetrating into the nanoporous film was measured using X-ray photoelectron spectroscopy (XPS). As presented in FIG. 4, the concentration of CFP-10 declined exponentially as a function of nanoporous film thickness and the majority of peptide accumulated within the top 100-nm layer. It was reasoned that it was not for lack of sufficient peptide holding space in the 196 nm films, but for the fact that the 643 nm thicker films provided additional reservoirs needed for the capillary-guided water flow and filling action that enhances the diffusion of CFP-10 within nanopore structures. Of all the nanopore configurations tested, the one resulting in an isolation efficiency up to 90% (36 ng of CFP-10) exhibited the following parameters: L121+25% PPG, 632-nm thickness, 7-mm ² surface area, and a 30-min incubation (FIG. 3B).

Adjusting the concentration of PPG not only affects pore size, but doing so also changes the structure's porosity, defined as the fraction of void spaces in the film. In FIG. 3B, comparable CFP-10 isolation efficiencies were observed when the L121 and L121+PPG films were used. However, other parameters were also considered that could singly, or collectively, improve the peptide enrichment and detection procedure, including the likely exclusion of abundant protein species in the sample, the efficiency of trypsin digestion, and sample elution. To test this hypothesis, recombinant CFP-10 was "spiked" into sterile MTB culture medium, the samples were processed on nanopore films of distinct characteristics, detected through MS, and then the MS signals of CFP-10 fragments were compared (FIG. 3C). To minimize the variation caused by the intrinsic fluctuations of MALDI MS, each extracted sample was spiked with isotopic peptides in known quantities to serve as internal standards. These isotopic peptides were synthesized by digested CFP-10 in ¹⁸ 0-enriched- water (H2 ¹⁸ 0), leading to their shift in mass by 4 Da without changing any other physical properties. Each MS signal shown in FIG. 3C was normalized by its own isotopic fragments. Although adjusting the pore size and porosity of L121 with PPG did not alter the amount of isolated CFP-10 (FIG. 3B), its impact became much more evident when the MS data were examined (FIG. 3C). Here, addition of PPG did have a positive effect on the detections of CFP-10 fragments, with the highest MS signals observed when the sample was processed on the L121+25% PPG nanoporous film. This increase tapers down and plateaus with further addition of PPG (100%). One possible reason for these observations is that the small pore size of L121 without PPG (avg. pore size: 3.9 nm) hinders the diffusion of globule-like trypsin (4 nm diameter), preventing interactions between trypsin and CFP-10 retained inside the nanotraps. With pore sizes beyond 5 nm, the effect of PPG on trypsin digestion was once again minimal to none (compare L121+25% PPG and L121+50% PPG to L121). Additionally, the larger pores retain more of the abundant proteins present in the sample, leading to a MS signal reduction of CFP-10 fragments. Indeed, we observed a significant decrease in MS signal intensity when the L121+100% PPG film (avg. pore size: 6.8 nm) was used.

Determining the Amount of CFP-10 from MTB Cultures

To quantify the absolute amount of CFP-10 fragments by their isotopic fragments, we first established a standard curve of the signal ratio of each monoisotopic and ¹⁸ 0- labeled fragment (FIG. 5). The isotopic fragments shifted by 4 Da to partially overlap with the mono-isotopic fragments. The fragments with 1142.63 and 1593.75([M+H]+) in MALDI TOF MS showed good linear regression between MS signal intensity and fragment quantity below 400nM (FIG. 5, R2 = 1.00 and 0.98, respectively), whereas the fragments of 1317.66 and 2003.98 ([M+H]+) demonstrated poor linear regression (R2 = 0.86 and 0.50, respectively). Based on standard curves for the fragments with 1 142.63 and 1593.75 ([M+H]+), the amount of CFP-10 was quantified after on-chip sample processing on different nanopore films. Similar to earlier results, CFP-10 processed on L121+25% PPG resulted in the highest yield at as 1.2 pmol (FIG. 3D).

To test the sensitivity of the assay, and determine its minimum threshold of detection, recombinant CFP-10 was titrated in sterile MTB culture medium, and each sample was processed on the L121+25% PPG film and through MS. The MS signals of four major fragments, each normalized to its own isotope, are depicted in FIG. 6B, plotted as signal intensity versus CFP-10 concentration in a log-log plot. Linear regression ranged from acceptable to good. Based on these results, this assay could detect CFP-10 in culture medium at a remarkably low concentration of 13.4 nM. The limit of detection was further improved (to 1.3 nM) by concentrating CFP-10 ten- fold by ammonium sulfate precipitation of the culture media before on-chip processing (FIG. 6C).

TABLE 2

INTER-DAY ACCURACY AND REPRODUCIBILITY OF CFP-10

ON-CHIP FRACTIONATION-MS ANALYSIS

Concentration Mean Standard Precision Accuracy

Fragments

g/mL) ^g/mL) Deviation (%CV) (%RE)

1317.664 1.7127 0.9940 58.03% 71.27%

1000

2003.978 0.0559 0.0351 62.66% 94.41%

1317.664 0.1361 0.0998 73.33% 8.91%

125

2003.978 0.0559 0.0351 62.66% 55.24%

1317.664 0.4283 0.4536 105.90% 2641.13%

15.625

2003.978 0.0031 0.0030 97.31% 80.20%

To access the inter-day and intra-day variability of the combined on-chip fractionation-MS analysis, recombinant CFP-10 was spiked at three different and defined concentrations, in replicate samples, into sterile culture media. The fragments with 1142.63 and 1593.75([M+H] ⁺ displayed higher MS signals (FIG. 1A, FIG. IB, and FIG. 1C), better linear regression with respect to their isotopes, and better quantification accuracy (%RE, relative error) and precision (% coefficient of variation, CV) compared to the fragments with 1317.66 and_2003.98 ([M+H] ⁺ ) (Table 2 and Table 3). At 100 nM concentration, the mean calculated concentrations remained within 10% of the actual values (%RE) and did not exceed 30% of the %CV. At lower concentrations, (1.3 nM) the accuracy of quantification decreased to 73%. The qualitative identification of CFP-10 remained very precise even at only 1.3 nM. Strong MS signals were detected for fragments with 1 142.63 and 1593.75([M+H] ⁺ ) in all of the samples (n = 1 1) and the fragments with 1317.66 and 2003.98 ([M+H] ⁺ ) in 63% and 72% of the samples. TABLE 3

INTRA-DAY ACCURACY AND REPRODUCIBILITY OF CFP-10

ON-CHIP FRACTIONATION-MS ANALYSIS

Concentration Mean Standard Precision Accuracy

Fragments

^g/mL) ^g/mL) Deviation (%CV) (%RE)

1317.664 1.2903 0.6716 52.05% 29.03%

1000

2003.978 0.0732 0.0412 56.32% 92.68%

146.544

1317.664 78.1665 53.34% 17.24%

125 6

2003.978 0.0732 0.0412 56.32% 41.44%

1317.664 0.4283 0.4536 105.90% 2641.13%

15.625

2003.978 0.0029 0.0033 112.94% 81.30%

Differentiating MTB Based on its CFP-10 Signatures in Clinical Isolates

To address specificity of the on-chip fractionation-MS technology, the expression of CFP-lOfrom MTB grown in culture medium was investigated (FIG. 7). The non-TB Mycobacterium (NTM), Mycobacterium avium (M. avium) lacks the CFP-10 gene, and therefore serves as a negative control. To mimic conditions one may find in early-disease diagnosis (i.e, low secretion of CFP-10 in the culture supernatant), the same ammonium sulfate concentration protocol was performed prior to on-chip fractionation and MS analysis. Indeed, strong MS signals for the all fragments were observed in the supernatant of MTB culture, but not in the M. avium culture (FIG. 7).

Sensitivity of Assay and Pre-Concentration ofESAT-6

To test the sensitivity of the current assay, on-chip fractionation and digestion process was applied to human serum and urine samples mixed with different concentrations of ESAT-6. The intensities of the 1901 Da fragment mass peak were measured by MALDI TOF MS at different concentrations, as shown in FIG. 8 and FIG. 13. 60 nM of ESAT-6 dissolved in either human serum (5%) or urine was still detectable using the nanopore-based assay described herein. To enhance the sensitivity of the assay, proteins within the patient sample were concentrated by precipitation before being applied onto sample on the surface of the nanoporous silica thin-film. As shown in FIG. 12, standard protein precipitation procedure increased ESAT-6 concentration in urine samples and enhanced the detection signal.

ELISA of ESAT-6- Comparative Example

Although enzyme-linked immunosorbent assay (ELISA) is often used to detect particular antigens in biological fluids, highly sensitive ELISAs (either sandwich or competitive ELISA), are not yet available. The indirect ELISA suffers from low sensitivity, especially in serum samples, because the analyte needs to compete with other abundant protein in biological fluids. The indirect ELISA was performed to detect ESAT-6 mixed in different biological fluids. As shown in FIG. 13, in the biological buffer without any other proteins, ESAT-6 signals were observed at 2 nM concentration. However, in human serum sample, ESAT-6 signals were not observed below micro-molar concentration. In contrast, the nanopore-based assay described herein sufficiently removed abundant proteins from complex body fluids, and thus enhanced the sensitivity of ESAT-6 detection in such body fluids.

Summary

This example provides a new sample pretreatment protocol for MALDI TOF MS using nanoporous silica film to fractionate and enrich ESAT-6. The on-chip fractionation and digestion protocols were established to provide fast diagnosis for TB screening. The silica nanopores were able to capture ESAT-6, to exclude abundant proteins that obscure the signals of target molecules in mass spectrometry, and to boost ESAT-6 fragment detection. The experimental results showed that pore sizes, porosity, and surface chemistry affect the captures capabilities of the nanoporous silica films. The methods described herein are also applicable for the identification of a range of other proteins of interest.

EXAMPLE 3 - SIMULTANEOUS DETECTION OF ESAT-6 AND CFP-10

To improve the sensitivity and specificity for TB diagnosis, both ESAT-6 and CFP- 10 were detected simultaneously from human body fluids. First, the finger printing spectra of ESAT-6 and CFP-10 were established (FIG. 14). Two major digestion fragments of ESAT-6 (molecular weights 1900.9511 and 1907.9246) were observed in the spectrum. Comparing to ESAT-6 fragments, more CFP-10 fragments were observed in MALDI TOF MS, including 2003.9781, 1668.7170, 1593.7503, 1 142.6276, and 908.4584 (Table 4). The most significant CFP-10 fragment (2003.9781) showed 10-fold higher in the intensity than the ESAT-6 fragment, which improved the sensitivity of this assay.

The human serum containing both 80 μΜ CFP-10 and 80 μΜ ESAT-6 antigens were fractionated with nanoporous silica films having a 6-nm average pore diameter, and then digested with trypsin. The standard protocol was then applied to process this sample as described above. MALDI TOF MS was then used to detect the antigen fragments in the eluted samples. The mass spectra showed that CFP-10 and ESAT-6 fragments were simultaneously observed (FIG. 15). CFP-10 showed higher intensity than ESAT-6. To improve the detection limit of nanoporous silica films in human serum, a 30-kDa cut-off ultracentrifugal filter was adopted for pre-concentration purposes. Then the pre- concentrated human serum with both CFP-10 and ESAT-6 was loaded and processed on nanoporous silica film (single-layer or dual-layer). As shown in FIG. 16, the detection limit of CFP-10 can reach as low as 100 nM in human serum after treated by both single-layer or dual-layer nanoporous silica film. Dual-layer nanoporous silica film showed less non- specific peaks than the single-layer one. Based on this result, it can be concluded that dual- layer nanoporous silica film increased the capture efficiency of target proteins compared to the single-layer one. After optimization, CFP-10 and ESAT-6 could be detected at concentrations less than 10 nM in human body fluid by coupling ultracentrifugal filtration with subsequent on-chip fractionation. Therefore, the assay described herein is useful to detect two or more antigens released from one or more infectious diseases. It offers significant improvements in sensitivity and specificity compared to existing assays, and has proven suitable for identifying multiple antigens/diseases in a single test.

TABLE 4

SEQUENCES AND MOLECULAR WEIGHTS OF CFP-10 AND ESAT-6 DIGESTED FRAGMENTS

[M+H]+ Sequence

CFP-10

2003.9781 TQIDQVESTAGSLQGQWR (SEQ ID NO:3)

1668.7170 ADEEQQQALSSQMGF (SEQ ID NO:4)

1593.7503 TDAATLAQEAGNFER (SEQ ID NO: 5)

1 142.6276 GAAGTAAQAAVVR (SEQ ID NO: 6)

908.4584 QAGVQYSR (SEQ ID NO:7)

ESAT-6

1907.9246 LAAAWGGSGSEAYQGVQQK (SEQ ID NO:2)

1900.951 1 WDATATELNNALQNLAR (SEQ ID NO: 1)

EXAMPLE 4 - FABRICATION OF DUAL-LAYER NANOPOROUS FILM

The dual layers of porous silica were fabricated using layer-by-layer coating recipe.

For an example shown in FIG. 17, the double layers silica film containing average 5 and 4 nm pore diameter on the top and bottom layers was fabricated by serially coating Pluronic L121 and Pluronic L121+25% PPG silicate sol on silicon wafers, according to the following protocol.

1. The different coating sols were prepared by mixing silicate sol with different type of Pluronic polymer. For 4 nm size pore, 10 mL of silicate sol was adding to the mixture of 1.2 g of Pluronic L121, and 5 mL of ethanol. For 5-nm size pore, 10 mL of silicate sol was added to a mixture of 1.2 g of Pluronic L121, 30 mL of ethanol, and 0.3 g of polypropylene glycol (PPG).

2. The coating solution was stirred at room temperature for 2 hr. The L121 coating sol was then deposited on a Si(100) wafer by spin coating at the spin rate of 500 rpm for 5 sec followed by spin coating at the spin rate of 2000 rpm for 20 sec. To increase the degree of polymerization of the silica framework in the films and to further improve their thermal stability, the as-deposited films were heated at 80°C for 12 hr. The films were calcinated firstly at 175°C for

3 hr and secondly at 450°C for 5 hr to remove the organic compound.

3. After the formation of first layer, the second layers were fabricated by depositing L121+25% PPG coating sol on the top of 4 nm pore size layer by spin coating at a spin rate of 500 rpm for 5 sec followed by spin coating at a spin rate of 2000 rpm for 20 sec. The films were first baked at 80°C for 12 hr, at 175°C for 3 hr, and then calcinated at 450°C for 5 hr to remove the organic compound.

Multilayer silica film can also be fabricated by this layer-by-layer coating recipe.

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All of the compositions and methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of exemplary embodiments, it will be apparent to those of ordinary skill in the art that variations may be applied to the composition, methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents that are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those of ordinary skill in the art are deemed to be within the spirit, scope and concept of the invention as defined herein.