Volatile activity-based nanosensors and uses thereof

Abstract

In some aspects, the disclosure relates to compositions and method for detection, classification, and treatment of disease or other body status. The methods and compositions may be methods or compositions for identification of diseases in patients using a nanosensor having an enzyme cleavage site linked to a volatile 5 reporter via a dipeptide linker.

Description

[0001] VOLATILE ACTIVITY-BASED NANOSENSORS AND USES THEREOF

[0002] RELATED APPLICATIONS

[0003] This application claims priority under 35 U. S. C. § 119(e) to U. S. Provisional Application, U. S. S. N. 63 / 739,745, filed December 30, 2024, which is incorporated herein by reference.

[0004] GOVERNMENT SUPPORT

[0005] This invention was made with Government support under Grant No. P30-CA14051 awarded by the National Cancer Institute, Grant No. P30-ES002109 awarded by the National Institute of Environmental Health Sciences, and NIH R01 Grant 5R01AI132413-07. The Government has certain rights in the invention.

[0006] REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0007] The contents of the electronic sequence listing (M065670555WO00-SEQ-FL.xml; Size: 46,199 bytes; and Date of Creation: December 17, 2025) are herein incorporated by reference in their entirety.

[0008] BACKGROUND

[0009] Breath biopsies can be used to link exhaled chemical signatures with specific medical conditions. For example, exhaled molecules including volatile organic compounds (VOCs) produced endogenously by metabolic processes throughout the body can be used as breath-based biomarkers for infectious diseases, cancer, asthma, and chronic obstructive pulmonary disease (COPD). However, creation of disease signatures is often challenging given heterogeneity in metabolome profiles, which are often influenced by patient’s physiology and environment. Therefore, efficient and specific methods and compositions for breath biopsies are warranted.

[0010] SUMMARY

[0011] To accelerate assessment of enzyme activity, disease detection, and disease monitoring, a diagnostic platform has been engineered that comprises multiplexable nanosensors that can be tuned to release volatile reporters into the breath in response to specific enzymes. In some instances the compositions disclosed herein leverage aberrant proteolytic activity for disease identification and / or disease monitoring via a breath test (FIG. 1).

[0012] Thus, in some aspects, the disclosure relates to methods and compositions for identification of diseases in patients using a nanosensor having an enzyme cleavage site linked

[0013] #14760451vlto a volatile reporter via a dipeptide linker. Without being bound by a particular theory, separation of the volatile reporter from the enzyme cleavage site using a dipeptide linker in a nanosensor allows for breath-based detection of endopeptidase activity. Aspects of the present disclosure provide nanosensors comprising a scaffold linked to an enzyme cleavage site, wherein the enzyme cleavage site is linked to a volatile reporter via a dipeptide linker.

[0014] In some embodiments, the enzyme cleavage site is a protease cleavage site.

[0015] In some embodiments, the protease cleavage site is cleaved by a protease, optionally wherein the protease is selected from trypsin, prolyl peptidase, a matrix metalloproteinase (MMP), a serine protease, granzyme B, cathepsin, and furin.

[0016] In some embodiments, the dipeptide linker comprises the amino acid sequence -Xaa'-Xp'-, wherein:

[0017] Xaa' is a natural amino acid or an unnatural amino acid (z.e., a compound that does not occur in nature but that can be incorporated into a peptide chain); and

[0018] Xp' is:

[0019] an optionally substituted heterocyclyl comprising at least 1 ring N atom, wherein the heterocyclyl is substituted with -CO2H and optionally one or more additional substituents; or

[0020] an optionally substituted heteroaryl comprising at least 1 ring N atom, wherein the heteroaryl is substituted with -CO2H and optionally one or more additional substituents.

[0021] In some embodiments, the dipeptide linker comprises the amino acid sequence -Xaa-Xp-, wherein:

[0022] Xaa is selected from glycine (Gly), alanine (Ala), serine (Ser), leucine (Leu), isoleucine (He), and phenylalanine (Phe); and

[0023] Xp is selected from proline (Pro), D-proline (D-pro), and a proline replacement.

[0024] In some embodiments, the proline replacement is of formula:

[0025]

[0026] °

[0027]

[0028] or a D-isomer thereof.

[0029] #14760451vlIn some embodiments, Xp is Pro, D-Pro, or a proline replacement, wherein the proline

[0030]

[0031] replacement is of formula: °, or a D-isomer thereof.

[0032] In some embodiments, the volatile reporter is a perfluorocarbon, an alcohol, or an amine, or an isotopically labeled derivative thereof, optionally wherein the isotopically labeled derivative of the alcohol is a deuterated alcohol, and optionally wherein the isotopically labeled derivative of the amine is a deuterated amine. In some embodiments, the volatile reporter is a perfluorocarbon or an alcohol, or an isotopically labeled derivative thereof, optionally wherein the isotopically labeled derivative of the alcohol is a deuterated alcohol.

[0033] In some embodiments, the perfluorocarbon is 2,2,3,3,3-pentafluoropropylamine (HFA1) or 1H,1H-perfluoropentylamine (HFA3).

[0034] In some embodiments, the alcohol is methanol, ethanol, 2-propanol, or 2-butanol, or the deuterated alcohol is ethanol-tZj, 2-propanol- z, or 2-butanol-J?.

[0035] Further aspects of the present disclosure provide compositions comprising one or more nanosensors disclosed herein.

[0036] Further aspects of the present disclosure provide compositions comprising two or more (e.g., three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, eleven or more, twelve or more, thirteen or more, fourteen or more, or fifteen or more) of the nanosensors described herein. In some embodiments, a composition comprises:

[0037] a first nanosensor comprising a scaffold linked to an enzyme cleavage site, wherein the enzyme cleavage site is linked to a volatile reporter via a dipeptide linker; and

[0038] a second nanosensor comprising a second scaffold linked to a second enzyme cleavage site, wherein a second volatile reporter is directly attached to the second enzyme cleavage site, optionally wherein:

[0039] the second scaffold is linked to the second enzyme cleavage site via a peptide, and the second volatile reporter is attached at a C-terminus of the peptide.

[0040] Further aspects of the present disclosure provide methods comprising one or more steps selected from:

[0041] (a) administering a nanosensor of the present disclosure to a subject;

[0042] (b) detecting in a breath sample obtained from a subject that has been administered a nanosensor of the present disclosure one or more volatile reporters that have been released from one or more nanosensors when exposed to an enzyme present in the subject;

[0043] #14760451vl(c) classifying a subject that has been administered a nanosensor of the present disclosure as having lung cancer; and

[0044] (d) classifying a subject that has been administered a nanosensor of the present disclosure as having a relapse of a lung cancer, optionally wherein the subject had previously received treatment for the lung cancer.

[0045] Each of the limitations of the invention can encompass various embodiments of the invention. It is, therefore, anticipated that each of the limitations of the invention involving any one element or combinations of elements can be included in each aspect of the invention. This invention is not limited in its application to the details of construction and the arrangement of components set forth in the following description or illustrated in the drawings. The invention is capable of other embodiments and of being practiced or of being carried out in various ways. Also, the phraseology and terminology used herein is for the purpose of description and should not be regarded as limiting. The use of “including,” “comprising,” or “having,” “containing”, “involving”, and variations thereof herein, is meant to encompass the items listed thereafter and equivalents thereof as well as additional items.

[0046] BRIEF DESCRIPTION OF DRAWINGS FIG. 1 shows development of multiplexed breath biomarkers for disease diagnostics. Top multiplexed volatile activity-based nanosensors (vABNs) are delivered locally to mouse lungs, where volatiles are produced, and exhaled breath is assayed by mass spectrometry.

[0047] Signatures of the detected volatile compounds were to train classifiers by machine learning algorithms that enable diagnosis of lung infection and lung cancer. Bottom: multiplexed vABNs comprise polyethylene glycol (PEG) nanoscaffolds coupled to volatile reporters through protease-cleavable linkers. Exopeptidase activities were recorded by amine volatiles through terminal cleavage, and endopeptidase activities by alcohol volatiles via controlled aminolysis. Schematic of mice was created in BioRender: Wang, S. (2024) BioRender.com / t96g624.

[0048] FIGs. 2A-2H show a mechanism of alcohol reporter generation. FIG. 2A shows a schematic showing protease cleavage site and substrate motifs to release alcohol reporters through aminolysis. Nt: N-terminus, Ct: C-terminus, P: proline. FIG. 2B shows a schematic of chemical-induced intramolecular aminolysis of dipeptide methyl ester. Anhydrous trifluoroacetic acid (TFA) was used to liberate the tert-butyloxycarbonyl (boc) group at the peptide N-terminus. Subsequent reconstitution in phosphate buffered saline (PBS) at 37 °C rapidly proceeds formation of diketopiperazine and methanol. FIG. 2C shows methanol generated from dipeptide glycine-proline methyl ester (GP-Ome) after chemical cleavage and reconstitution in PBS. FIGs.

[0049] #14760451vl2D-2E show kinetics of methanol produced by dipeptides with different Pl’ amino acids. FIG.

[0050] 2F shows chemical structures of P2’ proline analogs bearing different ring sizes and side chains. FIG. 2G shows percentage of methanol in the headspace generated by dipeptides with P2’ proline analogs 10 min after reconstitution in PBS. The maximum methanol produced was measured by the addition of tris buffer (50 mM, pH 7.5). FIG. 2H shows DFT analysis of the energy difference between cis and trans isomers. LP: 1-proline, DP: d-proline, Aze: azetidine, HP: homoproline, HyP: hydroxyproline, PhP: 4-phenylproline, BnP: 4-benzylproline.

[0051] FIGs. 3A-3H show volatile detection mediated by protease cleavage. FIG. 3A shows a schematic of a vABN where trypsin-sensing substrate S108 is attached to volatile reporters. The dotted line and * indicate the canonical cleavage site that initiates alcohol release by aminolysis. R indicates alkyl group. FIG. 3B shows MALDI-TOF spectra of S108 methyl ester (S108-Ome) before (top) and after incubation with human trypsin 3 (PRSS3, middle) or kallikrein-related peptidase 14 (KLK14, bottom) for 1 hr at 37 °C. FIGs. 3C-3D show methanol signal produced by S108-Ome with PRSS3 or KLK14. FIG. 3E shows methanol signal generated by S108(d-pro)-Ome with PRSS3, where P2’ is a d-proline. FIG. 3F shows S108(l-pro)-Ome and S108(d-pro)-Ome were used to assess non-specific cleavage by fibroblast activation protein (FAP). FIG.

[0052] 3G shows S108-Ome was used to assess PRSS3 activity modulated by solution pH. FIG. 3H shows Ethanol-< A signal generated by screening S108 ethyl-? ester (S108-Od5eth) against 16 recombinant human proteases associated with influenza A viral infection. Significance was calculated by two-tailed unpaired t tests (FIGs. 3C-3F) and two-way ANOVA with Bonferroni corrections for multiple comparisons (FIG. 3G); *P < 0.05, **P < 0.01.

[0053] FIGs. 4A-4I show development of multiplexed vABN in vivo. FIG. 4A shows a schematic of the influenza mouse model, where a single plex vABN was delivered 6 days postinfection. FIG. 4B shows mouse trypsin (PRSS3) protein levels were quantified by ELISA of BALF from healthy and PR8-infected mice collected at 2-, 4-, 6-, and 8-days post-infection. FIG. 4C shows trypsin-sensing vABN, S108-Ome, monitored progression of lung infection by detecting methanol released into BALF. FIG. 4D shows vABN-derived ethanol-^ levels were measured in exhaled breath of healthy and D6 PR8-infected mice at 10 min after S108 vABN delivery. FIG. 4E shows a schematic of an approach. Multiplexed vABNs, S70 (HFA1), S8 (HFA3), S108 (ethanol-^), S72 (2-propanol-60), and S26 (2-butanol- 3) were delivered to healthy and PR8 mice 6 days post-infection. FIG. 4F shows standardized volatile signals by z-scoring individual vABNs in the 5-plex panel, based on measurements in exhaled breath. FIG.

[0054] 4G shows a random forest classifier was applied to independent training and testing groups of healthy and PR8 mice (n=6 or 7 mice in each group). Performance was represented with an ROC

[0055] #14760451vlcurve and estimates of out-of-bag error was used for cross-validation. FIG. 4H shows distinguishing power of the trained classifier was visualized with a confusion matrix to differentiate healthy from PR8 mice. FIG. 41 shows a radar chart showing AUC values of individual vABNs in the multiplexed panel. Significance was calculated by one-way ANOVA with Bonferroni corrections for multiple comparisons (FIGs. 4B-4C) and two-tailed unpaired t tests (FIGs. 4D, 4F); *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

[0056] FIGs. 5A-5I show multiplexed vABNs for lung cancer detection. FIG. 5A shows a schematic of the Eml4-Alk (EA) model, where multiplexed vABNs were delivered at 4-, 5- or 6-weeks post tumor initiation. FIG. 5B shows a ROC curve representing the performance of a gradient boosting classifier in differentiating EA mice from healthy controls. The data is concatenated from two independent standardized cohort signals (healthy: n = 15, 16, 14 and EA: n = 25, 24, 21 for 4, 5, and 6 weeks, respectively). FIG. 5C shows a radar chart showing AUC values of individual vABNs in the multiplexed panel at different weeks. FIG. 5D shows a schematic of an approach. Multiplexed vABNs were delivered to mice one week after alectinib treatment and three weeks after discontinuing the treatment. FIG. 5E shows a violin plot summarizing the volatile signals of individual vABNs in the 5-plex panel among the three states of interest at week 6 post tumor initiation (healthy: n = 11, EA+vehicle: n = 14, EA+alectinib; n = 13). FIG. 5F shows PCA analysis was performed on standardized vABN signals from healthy controls and EA mice treated with either vehicle or alectinib. FIG. 5G shows representative microCT scans of representative lung tissues from mice after one week with or without alectinib exposure (W6), or three weeks after discontinuation of treatment (W10). Arrows point to tumors. FIG. 5H shows a ROC curve (top) and AUC values (bottom) show performance of a gradient boosting classifier in differentiating relapsed EA mice (n = 13) from healthy controls (n = 11) at W10. FIG. 51 shows relative individual vABN signals from a single cohort of mice at 6 weeks (with alectinib treatment) and at week 10 (three weeks after discontinuing the drug). The raw signals were normalized to the mean signals of healthy controls in the same cohort at the same week. Significance was calculated by one-way (FIG. 5E) or two-way (FIG. 51) ANOVAs with Bonferroni corrections for multiple comparisons; *P < 0.05, **P < 0.01, ***P < 0.001, **** P < 0.0001.

[0057] FIG. 6 shows that fluorescence kinetics show protease activity is Pl’ selective. Two quenched Anorogenic substrates S108-G (FAM-GAANLTRGP-CPQ2) (SEQ ID NO: 4) and S108-F (FAM-GAANLTRFP-CPQ2) (SEQ ID NO: 10) were designed for sensing trypsin-3 (PRSS3) activities, where Pl’ residue is either a glycine or a phenylalanine. Both substrates have a canonical cleavage site after Pl arginine. The substrates (5 pM) were mixed with PRSS3

[0058] #14760451vl(10 nM) in PBS in a 384-well plate (Corning). Fluorescence kinetics were measured at an excitation and emission of 485 nm and 535 nm, respectively, with a plate reader (Tecan Infinite 200 Pro M Plex) at 37 °C for 1 hr with an interval of 2 min. Fold change was calculated by fluorescence intensity at a given time to that at time = 0 min.

[0059] FIGs. 7A-7C show an analysis of aminolysis reaction mediated by chemical cleavage. Liquid chromatography-mass spectrometry (LC-MS) was used to analyze molecular weights of the reaction products in solution as described in FIG. 2B. (FIG. 7A) tert-butyloxycarbonyl (boc)-GP-OMe (100 ppm), (FIG. 7B) GP-Ome at time = 0 min upon reconstituting in water, and (FIG. 7C) GP-Ome after incubating in water for 1 hr at 37 °C. All samples were prepared in water and adjusted pH to 7. Molecular weight changes show deprotection of boc group by trifluoroacetic acid (FIG. 7B) and conversion to diketopiperazine (FIG. 7C).

[0060] FIG. 8 shows stability of boc-GP-Ome in various buffers. Boc-GP-Ome was incubated in 50 mM buffers for 1 hr at 37 °C prior to measuring methanol signal in the headspace. These selected buffers are commonly used in activity assays of recombinant proteases. Fold change was calculated by the signal ratios in buffers to water. Release of methanol was observed using tris buffer due to intermolecular aminolysis reaction between tris amine and the ester group of Boc-GP-Ome. Therefore, all tris buffers for protease activity assays were replaced with either HEPES or phosphate buffers.

[0061] FIGs. 9A-9G show an in vitro assessment of vABN substrates. FIG. 9A shows subsets of the urinary probes1for Eml4-Alk were re-engineered into vABN substrates. Their ability to differentiate healthy controls and Eml4-Alk mice were assessed using bronchoalveolar lavage fluids (BALF) collected from mice at 7 weeks after tumor initiation. The substrates contain methyl esters that can release methanol following hydrolysis at the canonical sites by target proteases (Tables 3A-3C). These methanol probes (100 pM) were incubated with BALF (0.1 mg / ml total proteins) overnight at 37 °C. Methanol signals in the headspace were measured by proton-transfer-reaction mass spectrometry (PTR-MS). Statistical significance was calculated by multiple two-sided t tests with the Holm-Sidak correction. FIG. 9B shows PP09 contains known MMP-cleavable motifs.2To identify its cleavage site by specific types of MMPs, two probes, PP09 (LP) and PP09 (LGP) were generated, which could release methanol following proteolytic cleavage at prescribed sites (dotted lines). FIGs. 9C-9D show methanol signal in the headspace after incubating 100 pM methanol probes with 100 nM recombinant human MMP9, MMP12, and MMP13 for 1 hour at 37 °C. These MMPs were elevated in the transcriptomic analyses of Eml4-Alk lungs1. FIGs. 9E-9G show ELIS As show MMP levels in BALF collected from healthy controls and Eml4-Alk mice at 7 weeks after tumor initiation.

[0062] #14760451vlFIGs. 10A-10E show volatile signals of vABN in representative multiplex panels.

[0063] Multiplexed vABNs were administered to healthy or Eml4-Alk mice at 4, 5 and 6 weeks after tumor initiation. Individual dots represent mouse in each cohort: 4 weeks (healthy n = 7, EA n = 15), 5 weeks (healthy n =8, EA n = 15), and 6 weeks (healthy n = 9, EA n = 15). The plots show raw signals of all time points collected (10, 30, 60 mins post administration) and standardized signal amplitudes integrated over 30 min by area under the curves; statistical analyses were performed respectively by two-way ANOVA with Bonferroni correction and two-tailed t tests in Prism 10.1.1. An additional dataset for each week was collected to perform machine learning classifications in FIG. 5B.

[0064] DETAILED DESCRIPTION

[0065] Breath biopsy is emerging as a rapid and non-invasive diagnostic tool that links exhaled chemical signatures with specific medical conditions. Despite its potential, clinical translation remains limited by the challenge of reliably detecting endogenous, disease-specific biomarkers in breath. In many instances, adaptation of nanosensors for breath biopsy has been constrained by the limited availability of orthogonal volatile reporters that are detectable in exhaled breath. Disclosed herein, in some embodiments, are engineered multiplexed breath biomarkers that couple aberrant protease activities to exogenous volatile reporters (<?.g., volatile reporters that are not endogenously produced). In some instances, the volatile reporters are synthetic volatile reporters (e.g., a volatile reporter may be a non-naturally occurring volatile reporter). In some instances, intramolecular reactions that leverage protease-mediated aminolysis are used herein to allow for the sensing of a broad spectrum of proteases, and that each release a unique reporter in breath. As shown in the Examples herein, this approach was used in a mouse model of influenza to establish baseline sensitivity and specificity in a controlled inflammatory setting, and subsequently applied to diagnose lung cancer using an autochthonous Aik- mutant model. By combining multiplexed reporter signals with machine learning algorithms, aspects of the present disclosure provide compositions and methods for tumor progression tracking, treatment response monitoring, and detection of relapse after 30 minutes. Without being bound by a particular theory, the multiplexed breath biopsy platform disclosed herein highlights a promising avenue for rapid, point-of-care diagnostics across diverse disease states.

[0066] Accessible and efficient diagnostics are needed for communicable and non-communicable diseases. Such tools may offer patients and their physicians the opportunity to personalize decision-making by allowing or early detection and longitudinal monitoring of both disease progression and treatment responses. Breath biopsy is a promising non-invasive

[0067] #14760451vldiagnostic approach alongside other minimally-invasive techniques, such as liquid biopsy, to facilitate personalized treatment and precision medicine without the need for tissue samples. Molecules produced endogenously by metabolic processes throughout the body, including volatile organic compounds (VOCs) and can serve as breath-based biomarkers for infectious diseases, cancer, asthma, and chronic obstructive pulmonary disease (COPD). However, identifying disease-specific VOC signatures remains a major challenge due to heterogeneity in the metabolome profiles, which can be highly influenced by an individual’s physiology and environment, resulting in variable breath VOC compositions.18

[0068] Catalytically-cleavable reporters designed to probe specific biological activity within diseased microenvironments can significantly enhance the signal-to-noise ratios of select analytes. Activity-based nanosensors (ABNs) can be used as diagnostic probes that comprise synthetic peptide substrates covalently coupled to exogenous reporters. These probes are often modular, such that the readout can be targeted to the activity of specific proteases, or classes of proteases, by changing the substrate sequence, and multiplexed signatures can be generated by incorporating distinguishable reporters. Upon protease cleavage of the ABNs, reporters are liberated from substrates and their relative concentrations in urine have been used to detect cancer, vascular disease, apoptosis, and inflammation. See, e.g., references 20-28 below.

[0069] Volatile-releasing activity-based nanosensors or volatile activity-based nanosensors (vABNs) are ABNs that generate a breath-based readout through the release of exogenous VOCs. vABNs were used to monitor neutrophil elastase activity in bacterial infection and alpha-1 antitrypsin deficiency, by releasing a single volatile fluorinated amine in the exhaled breath. See, e.g., WO2019173332 and Chan etal., Nat. Nanotechnol. 15, 792-800 (2020). However, without being bound by a particular theory, use of a terminal cleavage reaction to release the amine reporters in their native state could constrain both the target proteases that could be monitored using the vABN platform and the number of distinguishable volatiles that could be combined into a multiplexed panel. Without being bound by a particular theory, aspects of the present disclosure provide vABNs that use a reaction chemistry that could both respond to a broader range of protease classes and release a panel of orthogonal reporters with mass or chemical characteristics that distinguish them from endogenously produced VOCs, and each other.

[0070] Described herein, in some embodiments, are nanosensors with a specific mechanism for volatile release from a polymer core, whereby protease cleavage liberates a primary amine that attacks an ester in an intramolecular reaction to generate alcohol reporters (see, e.g., FIG. 1). Without being bound by a particular theory, this self-immolative reaction allows for selective

[0071] #14760451vlprobing of endopeptidase activity, which hydrolyzes non-terminal amino acids in a peptide substrate. Aspects of the present disclosure show that vABNs were generated to sense a broad spectrum of proteases that release volatile amines following exopeptidase cleavage at the terminus and volatile alcohols following cleavage by endopeptidases. This approach was demonstrated using a model of viral pneumonia to establish vABN baseline sensitivity and specificity in a controlled inflammatory setting, leveraging, e.g., well-characterized protease activities associated with viral entry, replication, and host responses.

[0072] The nanosensors of the present disclosure are modular so that volatile reporter release can be tuned to specific proteases by modification of the peptide linker sequence. In addition, peptide linkers may be replaced by other classes of substrates such as glycans, lipids, or nucleic acids to query glycosidase, lipase, and DNAse / RNAse activity, respectively. Different classes of volatile organic compounds may also be used in place of perfluorocarbon reporters. The interchangeability of the linker as well as reporter provides a number of possible nanosensor configurations for multiplexing. Aspects of the present disclosure provide a panel of five vABNs each comprising individual substrates that probe select protease activity and each release a unique amine or alcohol reporter that is detectable in exhaled breath. As demonstrated herein, multiplexed signals, measured by mass spectrometry and analyzed via machine learning algorithms accurately differentiated healthy and infected subjects.

[0073] As shown herein, this approach was also broadly applicable to chronic diseases, including lung cancer detection, assessment of treatment response, and detection of relapse. Lung cancer is the leading cause of global cancer mortality, with nearly 70% of patients diagnosed at advanced stages and a five-year survival rate of only 15%. The nanosensors described herein with volatile reporters were applied to an autochthonous mouse model of Aik mutant non- small-cell-lung cancer (NSCLC). As described herein, in some embodiments, a multiplexed panel of vABNs generated breath-based signals after 30 min of sensor delivery, and distinguished tumor-bearing mice from healthy controls, allowing for accurate assessment of treatment responses and post-treatment relapse. The multiplexed approach for breath biopsy along with the self-immolative chemistry could be used in a variety of settings, including diagnostics across various pathophysiological conditions.

[0074] Chemical Definitions

[0075] Definitions of specific functional groups and chemical terms are described in more detail below. The chemical elements are identified in accordance with the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75thEd., inside cover, and specific

[0076] #14760451vlfunctional groups are generally defined as described therein. Additionally, general principles of organic chemistry, as well as specific functional moieties and reactivity, are described in Thomas Sorrell, Organic Chemistry, University Science Books, Sausalito, 1999; Michael B. Smith, March’s Advanced Organic Chemistry, 7thEdition, John Wiley & Sons, Inc., New York, 2013; Richard C. Larock, Comprehensive Organic Transformations, John Wiley & Sons, Inc., New York, 2018; and Carruthers, Some Modern Methods of Organic Synthesis, 3rdEdition, Cambridge University Press, Cambridge, 1987.

[0077] Peptides and amino acids described herein can comprise one or more asymmetric centers, and thus can exist in various stereoisomeric forms, e.g., enantiomers and / or diastereomers. For example, the peptides described herein can be in the form of an individual enantiomer, diastereomer or geometric isomer, or can be in the form of a mixture of stereoisomers, including racemic mixtures and mixtures enriched in one or more stereoisomer. Isomers can be isolated from mixtures by methods known to those skilled in the art, including chiral high pressure liquid chromatography (HPLC) and the formation and crystallization of chiral salts; or preferred isomers can be prepared by asymmetric syntheses. See, for example, Jacques et al., Enantiomers, Racemates and Resolutions (Wiley Interscience, New York, 1981); Wilen et al., Tetrahedron 33:2725 (1977); Eliel, E. L. Stereochemistry of Carbon Compounds (McGraw-Hill, NY, 1962); and Wilen, S. H., Tables of Resolving Agents and Optical Resolutions p. 268 (E. L. Eliel, Ed., Univ, of Notre Dame Press, Notre Dame, IN 1972). The invention additionally encompasses peptides as individual isomers substantially free of other isomers, and alternatively, as mixtures of various isomers.

[0078] Unless otherwise provided, the disclosure includes peptides that do not include isotopically enriched atoms, and also includes peptides that include isotopically enriched atoms (“isotopically labeled derivatives”). For example, peptides having the present structures except for the replacement of hydrogen by deuterium or tritium, replacement of19F with18F, or the replacement of a carbon by a13C- or14C-enriched carbon are within the scope of the disclosure. The term “isotopes” refers to variants of a particular chemical element such that, while all isotopes of a given element share the same number of protons in each atom of the element, those isotopes differ in the number of neutrons.

[0079] When a range of values (“range”) is listed, it encompasses each value and sub-range within the range. A range is inclusive of the values at the two ends of the range unless otherwise provided. For example “Ci-6 alkyl” encompasses, Ci, C2, C3, C4, C5, Ce, Ci-6, C1-5, C1-4, C1-3, C1-2, C2-6, C2-5, C2-4, C2-3, C3-6, C3-5, C3-4, C4-6, C4-5, and C5-6 alkyl.

[0080] Use of the phrase “at least one instance” refers to 1, 2, 3, 4, or more instances, but also

[0081] #14760451vlencompasses a range, e.g., for example, from 1 to 4, from 1 to 3, from 1 to 2, from 2 to 4, from 2 to 3, or from 3 to 4 instances, inclusive.

[0082] A “non-hydrogen group” refers to any group that is defined for a particular variable that is not hydrogen.

[0083] The term “halo” or “halogen” refers to fluorine (fluoro, -F), chlorine (chloro, -Cl), bromine (bromo, -Br), or iodine (iodo, -I).

[0084] The term “alkyl” refers to a radical of a straight-chain or branched saturated hydrocarbon group having from 1 to 20 carbon atoms (“Ci-20 alkyl”). In some embodiments, an alkyl group has 1 to 6 carbon atoms (“Ci-6 alkyl”). Examples of Ci-6 alkyl groups include methyl (Ci), ethyl (C2), propyl (C3) (e.g., n-propyl, isopropyl), butyl (C4) (e.g., 77-butyl, tert-butyl, sec-butyl, isobutyl), pentyl (C5) (e.g., n-pentyl, 3-pentanyl, amyl, neopentyl, 3-methyl-2-butanyl, tertamyl), and hexyl (Cs) (e.g., n-hexyl). Additional examples of alkyl groups include n-heptyl (C7), 77-octyl (Cs), 77-dodecyl (C12), and the like.

[0085] The term “haloalkyl” is a substituted alkyl group, wherein one or more of the hydrogen atoms are independently replaced by a halogen, e.g., fluoro, bromo, chloro, or iodo.

[0086] “Fluoroalkyl” is a subset of haloalkyl, and refers to an alkyl group wherein one or more of the hydrogen atoms are independently replaced by a fluoro. “Perhaloalkyl” is a subset of haloalkyl, and refers to an alkyl group wherein all of the hydrogen atoms are independently replaced by a halogen, e.g., fluoro, bromo, chloro, or iodo. In some embodiments, the haloalkyl moiety has 1 to 20 carbon atoms (“Ci-20 haloalkyl”). In some embodiments, all of the haloalkyl hydrogen atoms are independently replaced with fluoro to provide a “perfluoroalkyl” group. Examples of haloalkyl groups include -CHF2, -CH2F, -CF3, -CH2CF3, -CF2CF3, -CF2CF2CF3, and the like.

[0087] The term “heteroalkyl” refers to an alkyl group, which further includes at least one heteroatom (e.g., 1, 2, 3, or 4 heteroatoms) selected from oxygen, nitrogen, or sulfur within (e.g., inserted between adjacent carbon atoms of) and / or placed at one or more terminal position(s) of the parent chain. In certain embodiments, a heteroalkyl group refers to a saturated group having from 1 to 20 carbon atoms and 1 or more heteroatoms within the parent chain (“heteroCi-20 alkyl”).

[0088] The term “alkenyl” refers to a radical of a straight-chain or branched hydrocarbon group having from 1 to 20 carbon atoms and one or more carbon-carbon double bonds (e.g., 1, 2, 3, or 4 double bonds). In some embodiments, an alkenyl group has 1 to 20 carbon atoms (“C1-20 alkenyl”). The one or more carbon-carbon double bonds can be internal (such as in 2-butenyl) or terminal (such as in 1-butenyl). In an alkenyl group, a C=C double bond for which the stereochemistry is not specified (e.g., -CH=CHCH3 or ) may be in the (£)- or (Z)-

[0089] #14760451vlconfiguration.

[0090] The term “heteroalkenyl” refers to an alkenyl group, which further includes at least one heteroatom (e.g., 1, 2, 3, or 4 heteroatoms) selected from oxygen, nitrogen, or sulfur within (e.g., inserted between adjacent carbon atoms of) and / or placed at one or more terminal position(s) of the parent chain. In certain embodiments, a heteroalkenyl group refers to a group having from 1 to 20 carbon atoms, at least one double bond, and 1 or more heteroatoms within the parent chain (“heteroCi-20 alkenyl”).

[0091] The term “alkynyl” refers to a radical of a straight-chain or branched hydrocarbon group having from 1 to 20 carbon atoms and one or more carbon-carbon triple bonds (e.g., 1, 2, 3, or 4 triple bonds) (“C1-20 alkynyl”). The one or more carbon-carbon triple bonds can be internal (such as in 2-butynyl) or terminal (such as in 1-butynyl).

[0092] The term “heteroalkynyl” refers to an alkynyl group, which further includes at least one heteroatom (e.g., 1, 2, 3, or 4 heteroatoms) selected from oxygen, nitrogen, or sulfur within (e.g., inserted between adjacent carbon atoms of) and / or placed at one or more terminal position(s) of the parent chain. In certain embodiments, a heteroalkynyl group refers to a group having from 1 to 20 carbon atoms, at least one triple bond, and 1 or more heteroatoms within the parent chain (“heteroCi-20 alkynyl”).

[0093] The term “carbocyclyl” or “carbocyclic” refers to a radical of a non-aromatic cyclic hydrocarbon group having from 3 to 14 ring carbon atoms (“C3-14 carbocyclyl”) and zero heteroatoms in the non-aromatic ring system. In some embodiments, a carbocyclyl group has 3 to 6 ring carbon atoms (“C3-6 carbocyclyl”). Exemplary C3-6 carbocyclyl groups include cyclopropyl (C3), cyclopropenyl (C3), cyclobutyl (C4), cyclobutenyl (C4), cyclopentyl (C5), cyclopentenyl (C5), cyclohexyl (Ce), cyclohexenyl (Ce), cyclohexadienyl (Ce), and the like. As the foregoing examples illustrate, in certain embodiments, the carbocyclyl group is either monocyclic (“monocyclic carbocyclyl”) or polycyclic (e.g., containing a fused, bridged or spiro ring system such as a bicyclic system (“bicyclic carbocyclyl”) or tricyclic system (“tricyclic carbocyclyl”)) and can be saturated or can contain one or more carbon-carbon double or triple bonds. “Carbocyclyl” also includes ring systems wherein the carbocyclyl ring, as defined above, is fused with one or more aryl or heteroaryl groups wherein the point of attachment is on the carbocyclyl ring, and in such instances, the number of carbons continue to designate the number of carbons in the carbocyclic ring system.

[0094] The term “heterocyclyl” or “heterocyclic” refers to a radical of a 3- to 14-membered non-aromatic ring system having ring carbon atoms and 1 to 4 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur (“3-14 membered

[0095] #14760451vlheterocyclyl”). In heterocyclyl groups that contain one or more nitrogen atoms, the point of attachment can be a carbon or nitrogen atom, as valency permits. In certain embodiments, the heterocyclyl is substituted or unsubstituted, 3- to 7-membered, monocyclic heterocyclyl, wherein 1, 2, or 3 atoms in the heterocyclic ring system are independently oxygen, nitrogen, or sulfur, as valency permits. A heterocyclyl group can either be monocyclic (“monocyclic heterocyclyl”) or polycyclic (e.g., a fused, bridged or spiro ring system such as a bicyclic system (“bicyclic heterocyclyl”) or tricyclic system (“tricyclic heterocyclyl”)), and can be saturated or can contain one or more carbon-carbon double or triple bonds. Heterocyclyl polycyclic ring systems can include one or more heteroatoms in one or both rings. “Heterocyclyl” also includes ring systems wherein the heterocyclyl ring, as defined above, is fused with one or more carbocyclyl groups wherein the point of attachment is either on the carbocyclyl or heterocyclyl ring, or ring systems wherein the heterocyclyl ring, as defined above, is fused with one or more aryl or heteroaryl groups, wherein the point of attachment is on the heterocyclyl ring, and in such instances, the number of ring members continue to designate the number of ring members in the heterocyclyl ring system.

[0096] The term “aryl” refers to a radical of a monocyclic or polycyclic (e.g., bicyclic or tricyclic) 4n+2 aromatic ring system (e.g., having 6, 10, or 14 pi electrons shared in a cyclic array) having 6-14 ring carbon atoms and zero heteroatoms provided in the aromatic ring system (“Ce-14 aryl”). In some embodiments, an aryl group has 6 ring carbon atoms (“Ce aryl”; e.g., phenyl). In some embodiments, an aryl group has 10 ring carbon atoms (“Cio aryl”; e.g., naphthyl such as 1-naphthyl and 2-naphthyl). In some embodiments, an aryl group has 14 ring carbon atoms (“Ci4 aryl”; e.g., anthracyl). “Aryl” also includes ring systems wherein the aryl ring, as defined above, is fused with one or more carbocyclyl or heterocyclyl groups wherein the radical or point of attachment is on the aryl ring, and in such instances, the number of carbon atoms continue to designate the number of carbon atoms in the aryl ring system.

[0097] The term “heteroaryl” refers to a radical of a 5-14 membered monocyclic or polycyclic (e.g., bicyclic, tricyclic) 4n+2 aromatic ring system (e.g., having 6, 10, or 14 pi electrons shared in a cyclic array) having ring carbon atoms and 1-4 ring heteroatoms provided in the aromatic ring system, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur (“5-14 membered heteroaryl”). In certain embodiments, the heteroaryl is substituted or unsubstituted, 5- or 6-membered, monocyclic heteroaryl, wherein 1, 2, 3, or 4 atoms in the heteroaryl ring system are independently oxygen, nitrogen, or sulfur. In certain embodiments, the heteroaryl is substituted or unsubstituted, 9- or 10-membered, bicyclic heteroaryl, wherein 1, 2, 3, or 4 atoms in the heteroaryl ring system are independently oxygen, nitrogen, or sulfur. In

[0098] #14760451vlheteroaryl groups that contain one or more nitrogen atoms, the point of attachment can be a carbon or nitrogen atom, as valency permits. Heteroaryl polycyclic ring systems can include one or more heteroatoms in one or both rings. “Heteroaryl” includes ring systems wherein the heteroaryl ring, as defined above, is fused with one or more carbocyclyl or heterocyclyl groups wherein the point of attachment is on the heteroaryl ring, and in such instances, the number of ring members continue to designate the number of ring members in the heteroaryl ring system. “Heteroaryl” also includes ring systems wherein the heteroaryl ring, as defined above, is fused with one or more aryl groups wherein the point of attachment is either on the aryl or heteroaryl ring, and in such instances, the number of ring members designates the number of ring members in the fused polycyclic (aryl / heteroaryl) ring system. Polycyclic heteroaryl groups wherein one ring does not contain a heteroatom (e.g., indolyl, quinolinyl, carbazolyl, and the like) the point of attachment can be on either ring, e.g., either the ring bearing a heteroatom or the ring that does not contain a heteroatom.

[0099] A chemical moiety is optionally substituted unless expressly provided otherwise. The term “optionally substituted” refers to being substituted or unsubstituted. In certain embodiments, alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, carbocyclyl, heterocyclyl, aryl, heteroaryl, acyl groups are optionally substituted. In general, the term “substituted” when referring to a chemical group means that at least one hydrogen present on the group is replaced with a permissible substituent, e.g., a substituent which upon substitution results in a stable compound, e.g., a compound which does not spontaneously undergo transformation such as by rearrangement, cyclization, elimination, or other reaction. Unless otherwise indicated, a “substituted” group has a substituent at one or more substitutable positions of the group, and when more than one position in any given structure is substituted, the substituent is either the same or different at each position. The invention is not limited in any manner by the exemplary substituents described herein.

[0100] Exemplary substituents include, but are not limited to, halogen, -CN, -NO2, -N3, -SO2H, -SO3H, -OH, -ORaa, -ON(Rbb)2, -N(Rbb)2, -N(Rbb)3+X“, -N(ORcc)Rbb, -SH, -SRaa, -SCN, -SSRCC, -C(=O)Raa, -CO2H, -CHO, -C(ORCC)2, -CO2Raa, -OC(=O)Raa, -OCO2Raa, -C(=O)N(Rbb)2, -OC(=O)N(Rbb)2, -NRbbC(=O)Raa, -NRbbCO2Raa, -NRbbC(=O)N(Rbb)2, -C(=NRbb)Raa, -C(=NRbb)ORaa, -OC(=NRbb)Raa, -OC(=NRbb)ORaa, -C(=NRbb)N(Rbb)2, -OC(=NRbb)N(Rbb)2, -NRbbC(=NRbb)N(Rbb)2, -C(=O)NRbbSO2Raa, -NRbbSO2Raa, -SO2N(Rbb)2, -SO2Raa, -SO2ORaa, -OSO2Raa, -S(=O)Raa, -OS(=O)Raa, -Si(Raa)3, -OSi(Raa)3-C(=S)N(Rbb)2, -C(=O)SRaa, -C(=S)SRaa, -SC(=S)SRaa, -SC(=O)SRaa, -OC(=O)SRaa, -SC(=O)ORaa, -SC(=O)Raa, -P(=O)(Raa)2, -P(=O)(ORCC)2, -OP(=O)(Raa)2, -OP(=O)(ORCC)2,

[0101] #14760451vl-P(=O)(N(Rbb)2)2, -OP(=O)(N(Rbb)2)2, -NRbbP(=O)(Raa)2, -NRbbP(=O)(ORcc)2, -NRbbP(=O)(N(Rbb)2)2, -P(RCC)2, -P(ORCC)2, -P(RCC)3+X“, -P(0RCC)3+X“, -P(RCC)4, -P(ORCC)4, -0P(RCC)2, -0P(RCC)3+X“, -0P(0RCC)2, -0P(0RCC)3+X-, -OP(RCC)4, -OP(ORCC)4, -B(Raa)2, -B(ORCC)2, -BRaa(ORcc), Ci-20 alkyl, Ci-20 perhaloalkyl, Ci-20 alkenyl, Ci-20 alkynyl, heteroCi-20 alkyl, heteroCi-20 alkenyl, heteroCi-20 alkynyl, C3-10 carbocyclyl, 3-14 membered heterocyclyl, Ce-14 aryl, and 5-14 membered heteroaryl; wherein X“ is a counterion;

[0102] or two geminal hydrogens on a carbon atom are replaced with the group =0, =S, =NN(Rbb)2, =NNRbbC(=O)Raa, =NNRbbC(=O)ORaa, =NNRbbS(=O)2Raa, =NRbb, or =NORCC; wherein:

[0103] each instance of Raais, independently, selected from Ci-20 alkyl, Ci-20 perhaloalkyl, Ci-20 alkenyl, Ci-20 alkynyl, heteroCi-20 alkyl, heteroCi-2oalkenyl, heteroCi-2oalkynyl, C3-10 carbocyclyl, 3-14 membered heterocyclyl, Ce-14 aryl, and 5-14 membered heteroaryl, or two Raagroups are joined to form a 3-14 membered heterocyclyl or 5-14 membered heteroaryl ring; each instance of Rbbis, independently, selected from hydrogen, -OH, -0Raa, -N(RCC)2, -CN, -C(=O)Raa, -C(=O)N(RCC)2, -CO2Raa, -SO2Raa, -C(=NRcc)0Raa, -C(=NRCC)N(RCC)2, -SO2N(RCC)2, -SO2RCC, -SO2ORCC, -SOR^, -C(=S)N(RCC)2, -C(=O)SRCC, -C(=S)SRCC, -P(=O)(Raa)2, -P(=O)(ORCC)2, -P(=O)(N(RCC)2)2, CI-20 alkyl, Ci-20 perhaloalkyl, Ci-20 alkenyl, C1-20 alkynyl, heteroCi-2oalkyl, heteroCi-2oalkenyl, heteroCi-2oalkynyl, C3-10 carbocyclyl, 3-14 membered heterocyclyl, Ce-14 aryl, and 5-14 membered heteroaryl, or two Rbbgroups are joined to form a 3-14 membered heterocyclyl or 5-14 membered heteroaryl ring;

[0104] each instance of Rccis, independently, selected from hydrogen, Ci-20 alkyl, Ci-20 perhaloalkyl, Ci-20 alkenyl, Ci-20 alkynyl, heteroCi-20 alkyl, heteroCi-20 alkenyl, heteroCi-20 alkynyl, C3-10 carbocyclyl, 3-14 membered heterocyclyl, Ce-14 aryl, and 5-14 membered heteroaryl, or two Rccgroups are joined to form a 3-14 membered heterocyclyl or 5-14 membered heteroaryl ring; and each X“ is a counterion.

[0105] Affixing the suffix “-ene” to a group indicates the group is a divalent moiety, e.g., alkylene is the divalent moiety of alkyl, alkenylene is the divalent moiety of alkenyl, alkynylene is the divalent moiety of alkynyl, heteroalkylene is the divalent moiety of heteroalkyl, heteroalkenylene is the divalent moiety of heteroalkenyl, heteroalkynylene is the divalent moiety of heteroalkynyl, carbocyclylene is the divalent moiety of carbocyclyl, heterocyclylene is the divalent moiety of heterocyclyl, arylene is the divalent moiety of aryl, and heteroarylene is the divalent moiety of heteroaryl.

[0106] Linkers

[0107] #14760451vlAs used herein “linked” or “linkage” means two entities are bound to one another by any physicochemical means. Any linkage known to those of ordinary skill in the art, covalent or non-covalent, is embraced. Examples of linking molecules include but are not limited to polyethylene glycol), peptide linkers, N-(2-Hydroxypropyl) methacrylamide linkers, elastinlike polymer linkers, and other polymeric linkages. Generally, a linking molecule is a polymer and may comprise between about 2 and 200 (e.g., any integer between 2 and 200, inclusive) molecules.

[0108] In some embodiments, a linking molecule comprises one or more poly(ethylene glycol) (PEG) molecules. In some embodiments, a linking molecule comprises between 2 and 200 (e.g., any integer between 2 and 200, inclusive) PEG molecules. In some embodiments, a linking molecule comprises between 2 and 20 PEG molecules. In some embodiments, a linking molecule comprises between 5 and 15 PEG molecules. In some embodiments, a linking molecule comprises between 5 and 25 PEG molecules. In some embodiments, a linking molecule comprises between 10 and 40 PEG molecules. In some embodiments, a linking molecule comprises between 25 and 50 PEG molecules. In some embodiments, a linking molecule comprises between 100 and 200 PEG molecules.

[0109] In some embodiments, two molecules are linked using a transpeptidase, for example Sortase A.

[0110] Dipeptide Linkers

[0111] The term “amino acid” refers to a molecule containing both an amino group and a carboxyl group. Unless otherwise indicated, an amino acid is an alpha-amino acid (a-amino acid), the generic structure of which is depicted below (wherein each R is independently H or an amino acid sidechain, i.e., an “a- sidechain”). Unless otherwise indicated, reference to a particular amino acid implies the L-isomer of the amino acid. Each amino acid referred to herein may be denoted by a 1- to 4-letter code.

[0112]

[0113] O

[0114] a-amino acid

[0115] Suitable amino acids include, without limitation, natural a-amino acids such as D- and L-isomers of the 20 common naturally occurring a-amino acids found in peptides e.g., A, R, N, C, D, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y, V, as provided below), and unnatural a-amino acids.

[0116] Exemplary natural a-amino acids (with one-letter code provided in parentheses) include

[0117] #14760451vlL-alanine (A), L-arginine (R), L-asparagine (N), L-aspartic acid (D), L-cysteine (C), L-glutamic acid (E), L-glutamine (Q), glycine (G), L-histidine (H), L-isoleucine (I), L-leucine (L), L-lysine (K), L-methionine (M), L-phenylalanine (F), L-proline (P), L-serine (S), L-threonine (T), L-tryptophan (W), L-tyrosine (Y), and L- valine (V).

[0118] Exemplary unnatural a-amino acids include, without limitation, D-arginine, D-asparagine, D-aspartic acid, D-cysteine, D-glutamic acid, D-glutamine, D-histidine, D-isoleucine, D-leucine, D-lysine, D-methionine, D-phenylalanine, D-proline, D- serine, D-threonine, D-tryptophan, D-tyrosine, D-valine, Di- vinyl, a-methyl-alanine (Aib), a-methyl-arginine, a-methyl-asparagine, a-methyl-aspartic acid, a-methyl-cysteine, a-methyl-glutamic acid, a-methyl-glutamine, a-methyl-histidine, a-methyl-isoleucine, a-methyl-leucine, a-methyl-lysine, a-methyl-methionine, a-methyl-phenylalanine, a-methyl-proline, a- methyl- serine, a-methyl-threonine, a-methyl-tryptophan, a-methyl-tyrosine, a-methyl-valine, norleucine, and terminally unsaturated a-amino acids. There are many known unnatural amino acids, any of which may be included in the peptides of the present disclosure. See for example, S. Hunt, The Non-P rotein Amino Acids: In Chemistry and Biochemistry of the Amino Acids, edited by G. C. Barrett, Chapman and Hall, 1985.

[0119] The term “peptide” refers to a polymer of amino acid residues linked together by peptide bonds. Typically, a peptide will be at least two amino acids long, or at least the length required by an amino acid sequence provided herein. Peptides provided herein can include natural amino acids and / or unnatural amino acids (z.e., compounds that do not occur in nature but that can be incorporated into a peptide chain) in any combination. One or more of the amino acids in a peptide may be modified, for example, by the addition of a chemical entity such as a carbohydrate group, a hydroxyl group, a phosphate group, a farnesyl group, an isofamesyl group, a fatty acid group, a linker for conjugation or functionalization, or other modification. A peptide may be naturally occurring, recombinant, synthetic, or any combination of these.

[0120] The term “dipeptide” refers to two amino acid residues linked together by a peptide bond. A dipeptide described herein can include natural amino acids and / or unnatural amino acids (z.e., compounds that do not occur in nature but that can be incorporated into a peptide chain) in any combination. As a non-limiting example, the dipeptide Gly-Pro (glycine-proline, GP) has the formula:

[0121]

[0122] A peptide (e.g., a dipeptide) can comprise one or more additional modifications. For

[0123] #14760451vlexample, in some embodiments, the N-terminus of the peptide is acetylated (denoted by “Ac-” at the N-terminus of an amino acid sequence) or tert-butyloxycarbonyl (Boc)-protected (denoted by “Boc-” at the N-terminus of an amino acid sequence). In some embodiments, the C-terminus of the peptide is esterified, i.e., the traditional C-terminal -C(=O)OH is replaced with -C(=O)ORc, wherein Rcis a non-hydrogen moiety, or amidated, i.e., the traditional C-terminal -C(=O)OH is replaced with -C(=0)NH2 or -C(=O)NHRN, wherein RNis a non-hydrogen moiety. In some embodiments, a peptide provided herein comprises an acetylated or Boc-protected N-terminus and an esterified or amidated C-terminus. As a non-limiting example, the tertbutyloxycarbonyl (boc)-protected Gly-Pro methyl ester (Boc-protected Gly-Pro-Ome, Boc-Gly-Pro-Ome) has the formula:

[0124] N

[0125] z ~N

[0126]

[0127] O H

[0128] As generally described herein, a nanosensor of the present disclosure comprises a scaffold linked to an enzyme cleavage site, wherein the enzyme cleavage site is linked to a volatile reporter via a dipeptide linker.

[0129] The term “dipeptide linker” refers to a divalent dipeptide moiety (i.e., a dipeptidylene group). As a non-limiting example, the dipeptide linker -Gly-Pro- has the formula:

[0130]

[0131] In some embodiments, the dipeptide linker comprises the amino acid sequence -Xaa'-Xp'-, wherein:

[0132] Xaa' is a natural amino acid or an unnatural amino acid (i.e., a compound that does not occur in nature but that can be incorporated into a peptide chain); and

[0133] Xp' is:

[0134] an optionally substituted heterocyclyl comprising at least 1 ring N atom, wherein the heterocyclyl is substituted with -CO2H and optionally one or more additional substituents; or

[0135] an optionally substituted heteroaryl comprising at least 1 ring N atom, wherein the heteroaryl is substituted with -CO2H and optionally one or more additional substituents.

[0136] In some embodiments, the dipeptide linker comprises the amino acid sequence -Xaa-Xp-, wherein:

[0137] #14760451vlXaa is selected from glycine (Gly), alanine (Ala), serine (Ser), leucine (Leu), isoleucine (He), and phenylalanine (Phe); and

[0138] Xp is selected from proline (Pro), D-proline (D-pro), and a proline replacement.

[0139] In some embodiments, Xp is Pro. In some embodiments, Xp is D-Pro. In some embodiments, Xaa is Gly and Xp is Pro or D-Pro.

[0140] As described herein, “proline replacement” refers to an amino acid of Formula (a):

[0141]

[0142] or a D-isomer thereof, wherein:

[0143] pl is 0, 1, 2, or 3;

[0144] each R4is independently halogen, Ci-6 alkyl, Ci-6 haloalkyl, -CN, -ORA, -N(RA)2, -SRA, -C(=O)RA, -C(=O)ORA, -C(=O)N(RA)2, C3-8 carbocyclyl, Ce-ioaryl, 3-8 membered heterocyclyl, or 5-10 membered heteroaryl, or two R4attached to the same carbon atom are joined together with the intervening atoms to form C3-8 carbocyclyl or 3-8 membered heterocyclyl, or two R4are taken together to form =0; and

[0145] each instance of RAis independently hydrogen, C1-6 alkyl, C1-6 haloalkyl, C3-8 carbocyclyl, Ce-ioaryl, 3-8 membered heterocyclyl, or 5-10 membered heteroaryl, or two RAattached to the same nitrogen atom are joined together to form 3-8 membered heterocyclyl; and p2 is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, as valency permits.

[0146] In some embodiments, the proline replacement is of formula:

[0147]

[0148] °

[0149]

[0150] or a D-isomer thereof.

[0151] In some embodiments, Xp is Pro, D-Pro, or a proline replacement, wherein the proline

[0152] replacement is of formula:

[0153]

[0154] or a D-isomer thereof.

[0155] #14760451vlVolatile reporters

[0156] As used herein, a volatile reporter is capable of vaporizing at room temperature. In some instances, a volatile reporter is capable of partitioning from liquid phase into headspace. In some instances, a volatile reporter is capable of phase transition from liquid to gas. A volatile reporter may comprise a volatile organic compound (VOC). A volatile reporter may be naturally produced by a cell or subject and may be referred to as an endogenous volatile reporter. In some instances, a volatile reporter is not naturally produced by a cell or organism. As used herein, a synthetic volatile reporter is a volatile reporter that is not naturally produced by a cell or a subject. In some instances, the subject is a human.

[0157] A volatile reporter may comprise at least one 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, or at least 100 volatile organic compounds. In some instances, the volatile organic compound is a perfluorocarbon.

[0158] As a non-limiting example, a volatile reporter may comprise a perfluorocarbon (PFC). PFCs are fluorinated carbon compounds. In some instances, a PFC comprises a carbonfluorine bond. In some embodiments, a PFC comprising is a perfluoroalkane.

[0159] There are at least four types of PFCs. In some instances, PFCs are cyclic, branched, or linear, completely fluorinated alkanes; cyclic, branched, or linear, completed fluorinated ethers, with no unsaturations; cyclic, branched, or linear, completely fluorinated tertiary-amines with no unsaturations; or sulfur containing perfluorocarbons with no unsaturations and with sulfur bonds only to carbon and fluorine). In some embodiments, the perfluorocarbon has the chemical formula CF3(CF2)XCH2NH2. In some instances, x in the chemical formula CF3(CF2)XCH2NH2 is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, or 100, including any values in between. In some instances, the perfluorocarbon is 2, 2, 3,3,3-pentafluoropropylamine (HFA1) or 1H,1H-perfluoropentylamine (HFA3). In some instances, the VOCs are biocompatible and highly volatile (have a high vapor pressure).

[0160] Unless otherwise provided, the disclosure includes volatile reporters that do not include isotopically enriched atoms, and also includes volatile reporters that include isotopically enriched atoms (“isotopically labeled derivatives”). For example, volatile reporters having the present structures except for the replacement of hydrogen by deuterium or tritium, replacement of19F with18F, or the replacement of a carbon by a13C- or14C-enriched carbon are within the scope of the disclosure.

[0161] In some embodiments, the volatile reporter is an alcohol, or an isotopically labeled

[0162] #14760451vlderivative thereof. In some embodiments, the alcohol is methanol, ethanol, 1 -propanol, 2-propanol, 1 -butanol, 2-butanol, isobutanol, or tert-butanol. In some embodiments, the alcohol is methanol, ethanol, 2-propanol, or 2-butanol.

[0163] In some embodiments, the volatile reporter is an isotopically labeled derivative of an alcohol. In some embodiments, the alcohol is isotopically enriched in deuterium. In some embodiments, the isotopically labeled derivative of the alcohol is a deuterated alcohol. In some embodiments, the deuterated alcohol is an isotopically labeled derivative of methanol, ethanol, 1 -propanol, 2-propanol, 1 -butanol, 2-butanol, isobutanol, or tert-butanol. In some embodiments, the deuterated alcohol is ethanol-, 2-propanoI- z, or 2-butanoI-?.

[0164] In some embodiments, the volatile reporter is an amine, or an isotopically labeled derivative thereof. In some embodiments, the amine is a primary amine, a secondary amine, or a tertiary amine. In some embodiments, the amine is methylamine, ethylamine, zz-propylamine, isopropylamine, -butylamine, ec-butylamine, isobutylamine, or tert-butylamine.

[0165] In some embodiments, the volatile reporter is an isotopically labeled derivative of an amine. In some embodiments, the amine is isotopically enriched in deuterium. In some embodiments, the isotopically labeled derivative of the amine is a deuterated amine. In some embodiments, the deuterated amine is an isotopically labeled derivative of a primary amine, a secondary amine, or a tertiary amine. In some embodiments, the deuterated amine is an isotopically labeled derivative of methylamine, ethylamine, n-propylamine, isopropylamine, n-butylamine, ec-butylamine, isobutylamine, or tert-butylamine.

[0166] In some instances, the volatile organic compounds (VOCs) comprise an amine. In some instances the amine is useful for conjugation to a scaffold.

[0167] Additional classes of VOCs include food flavorings. For example, the food flavorings may comprise an alcohol or a thiol. In some instances, a VOC is methanethiol, 2-Propene-l-thiol, 2-Proanethiol, 1-ropanethiol, 2-Methyl-l -propanethiol, 1 -Butanethiol, 2-Pentanethiol, 3-Methyl-1 -butanethiol, 1 -Pentanethiol, ethanol, propanol, isopropyl alcohol, isobutyl alcohol, butyl alcohol, 2-Methyl-3-buten-2-ol, l-Penten-3-ol, Isoamyl alcohol, or amyl alcohol. In some instances, a VOC may comprise a sulfur, hydroxyl group, or any combination thereof and have a high vapor pressure and a low boiling point. In some instances, the VOCs are not bio-orthogonal and may have naturally produced metabolite counterparts. In contrast, perfluorocarbons and deuterated alcohols may be considered bio-orthogonal, as they are not naturally produced in the human body.

[0168] Any suitable method known in the art or described herein may be used to detect a volatile reporter. For example, a VOC in a volatile reporter may be detected and detection of a

[0169] #14760451vlVOC may comprise gas chromatography, mass spectrometry, gas chromatography-mass spectrometry (GC-MS), chemiluminescence, use of electronic noses, optical absorption spectroscopy, ion mobility spectroscopy, use of different types of gaseous sensors, or any combination thereof. See, e.g., Sethi et al., Clin Microbiol Rev. 2013 Jul;26(3):462-75.

[0170] Scaffolds

[0171] The enzyme nanosensor comprises a modular structure having a scaffold linked to an enzyme- specific substrate. The enzyme- specific substrate may comprise an enzyme cleavage site. For example, the enzyme cleavage site may be a protease cleavage site. A modular structure, as used herein, refers to a molecule having multiple domains.

[0172] The scaffold may be linked to a single type of substrate, such as, a single type of enzyme- specific substrate, or it may include multiple types of different substrates. For instance each scaffold may be linked to a single (e.g., 1) type of substrate or it may be linked to 2-1,000 different substrates, or any integer therebetween. Alternatively, each scaffold may be linked to greater than 1,000 different substrates. Multiple copies of the enzyme nanosensor are administered to the subject. In some embodiments, a composition comprising a plurality of different nanosensors may be administered to a subject to determine whether multiple enzymes and / or substrates are present. In that instance, the plurality of different nanosensors includes a plurality of volatile reporters, such that each substrate is associated with a particular volatile reporter.

[0173] The scaffold may serve as the core of the nanosensor. A purpose of the scaffold is to serve as a platform for the substrate and enhance delivery of the nanosensor to the subject. As such, the scaffold can be any material or size as long as it can enhance delivery and / or accumulation of the nanosensors to the subject. Preferably, the scaffold material is non-immunogenic, i.e. does not provoke an immune response in the body of the subject to which it will be administered. Non-limiting examples of scaffolds, include, for instance, compounds that cause active targeting to tissue, cells or molecules, microparticles, nanoparticles, aptamers, peptides (RGD, iRGD, LyP-1, CREKA, etc.), proteins, nucleic acids, polysaccharides, polymers, antibodies or antibody fragments (e.g., herceptin, cetuximab, panitumumab, etc.) and small molecules (e.g., erlotinib, gefitinib, sorafenib, etc.).

[0174] Without being bound by a particular theory, in some embodiments, delivery of a nanosensor described herein to a subject by inhalation is enhanced by nanosensors having certain polymer scaffolds (e.g., polyethylene glycol) (PEG) scaffolds). Polyethylene glycol (PEG), also known as poly(oxyethylene) glycol, is a condensation polymer of ethylene oxide

[0175] #14760451vland water having the general chemical formula HO(CH2CH2O)[n]H. Generally, a PEG polymer can range in size from about 2 subunits (e.g., ethylene oxide molecules) to about 50,000 subunits (e.g., ethylene oxide molecules. In some embodiments, a PEG polymer comprises between 2 and 10,000 subunits (e.g., ethylene oxide molecules).

[0176] A PEG polymer can be linear or multi-armed (e.g., dendrimeric, branched geometry, star geometry, etc.). In some embodiments, a scaffold comprises a linear PEG polymer. In some embodiments, a scaffold comprises a multi-arm PEG polymer. In some embodiments, a multiarm PEG polymer comprises between 2 and 20 arms. In some embodiments, a multi-arm PEG polymer comprises 8 arms. In some embodiments, a multi-arm PEG is an 8-arm 40 kDa PEG. Multi-arm and dendrimeric scaffolds are generally described, for example by Madaan et al. J Pharm Bioallied Sci. 2014 6(3): 139-150.

[0177] Additional polymers include, but are not limited to: polyamides, polycarbonates, polyalkylenes, polyalkylene glycols, polyalkylene oxides, polyalkylene terepthalates, polyvinyl alcohols, polyvinyl ethers, polyvinyl esters, polyvinyl halides, polyglycolides, polysiloxanes, polyurethanes and copolymers thereof, alkyl cellulose, hydroxyalkyl celluloses, cellulose ethers, cellulose esters, nitro celluloses, polymers of acrylic and methacrylic esters, methyl cellulose, ethyl cellulose, hydroxypropyl cellulose, hydroxy-propyl methyl cellulose, hydroxybutyl methyl cellulose, cellulose acetate, cellulose propionate, cellulose acetate butyrate, cellulose acetate phthalate, carboxylethyl cellulose, cellulose triacetate, cellulose sulphate sodium salt, poly(methyl methacrylate), poly (ethylmethacrylate), poly (butylmethacrylate),

[0178] poly (isobutylmethacrylate), poly(hexlmethacrylate), poly(isodecylmethacrylate), poly(lauryl methacrylate), poly(phenyl methacrylate), poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutyl acrylate), poly(octadecyl acrylate), polyethylene, polypropylene polyethylene glycol), polyethylene oxide), poly(ethylene terephthalate), poly(vinyl alcohols), poly(vinyl acetate, poly vinyl chloride and polystyrene.

[0179] Examples of non-biodegradable polymers include ethylene vinyl acetate, poly(meth) acrylic acid, polyamides, copolymers and mixtures thereof.

[0180] Examples of biodegradable polymers include synthetic polymers such as polymers of lactic acid and glycolic acid, polyanhydrides, poly(ortho)esters, polyurethanes, poly(butic acid), poly(valeric acid), poly(caprolactone), poly (hydroxybutyrate), poly(lactide-co-glycolide) and poly(lactide-co-caprolactone), and natural polymers such as algninate and other polysaccharides including dextran and cellulose, collagen, chemical derivatives thereof (substitutions, additions of chemical groups, for example, alkyl, alkylene, hydroxylations, oxidations, and other modifications routinely made by those skilled in the art), albumin and other hydrophilic proteins,

[0181] #14760451vlzein and other prolamines and hydrophobic proteins, copolymers and mixtures thereof. In general, these materials degrade either by enzymatic hydrolysis or exposure to water in vivo, by surface or bulk erosion. The foregoing materials may be used alone, as physical mixtures (blends), or as co-polymers. In some embodiments the polymers are polyesters, polyanhydrides, polystyrenes, polylactic acid, polyglycolic acid, and copolymers of lactic and glycoloic acid and blends thereof.

[0182] PVP is a non-ionogenic, hydrophilic polymer having a mean molecular weight ranging from approximately 10,000 to 700,000 and the chemical formula (C6H9NO)n. PVP is also known as poly[l-(2-oxo-l -pyrrolidinyl)ethylene], POVIDONE™, POLYVIDONE™, RP 143™, KOLLIDON™, PEREGAL ST™, PERISTON™, PLASDONE™, PLASMOSAN™, PROTAGENT™, SUBTOSAN™, and VINISIL™. PVP is non-toxic, highly hygroscopic and readily dissolves in water or organic solvents.

[0183] Polyvinyl alcohol (PVA) is a polymer prepared from polyvinyl acetates by replacement of the acetate groups with hydroxyl groups and has the formula (CH2CHOH)[n], Most polyvinyl alcohols are soluble in water.

[0184] PEG, PVA and PVP are commercially available from chemical suppliers such as the Sigma Chemical Company (St. Louis, Mo.).

[0185] In certain embodiments the particles may comprise poly(lactic-co-glycolic acid) (PLGA). In some embodiments, a scaffold (e.g., a polymer scaffold, such as a PEG scaffold) has a molecular weight equal to or greater than 40 kDa. In some embodiments, a scaffold is a nanoparticle (e.g., an iron oxide nanoparticle, IONP) that is between 10 nm and 50 nm in diameter (e.g. having an average particle size between 10 nm and 50 nm, inclusive). In some embodiments, a scaffold is a high molecular weight protein, for example an Fc domain of an antibody.

[0186] In some embodiments, a scaffold comprises a particle. As used herein the term “particle” includes nanoparticles as well as microparticles. Nanoparticles are defined as particles of less than 1.0 pm in diameter. A preparation of nanoparticles includes particles having an average particle size of less than 1.0 pm in diameter. Microparticles are particles of greater than 1.0 pm in diameter but less than 1 mm. A preparation of microparticles includes particles having an average particle size of greater than 1.0 pm in diameter. The microparticles may therefore have a diameter of at least 5, at least 10, at least 25, at least 50, or at least 75 microns, including sizes in ranges of 5-10 microns, 5-15 microns, 5-20 microns, 5-30 microns, 5-40 microns, or 5-50 microns. A composition of particles may have heterogeneous size distributions ranging from 10 nm to mm sizes. In some embodiments the diameter is about 5 nm

[0187] #14760451vlto about 500 nm. In other embodiments, the diameter is about 100 nm to about 200 nm. In other embodiment, the diameter is about 10 nm to about 100 nm.

[0188] The particles may be composed of a variety of materials including iron, ceramic, metallic, natural polymer materials (including lipids, sugars, chitosan, hyaluronic acid, etc.), synthetic polymer materials (including poly-lactide-coglycolide, poly-glycerol sebacate, etc.), and non-polymer materials, or combinations thereof.

[0189] The particles may be composed in whole or in part of polymers or non-polymer materials. Non-polymer materials, for example, may be employed in the preparation of the particles. Exemplary materials include alumina, calcium carbonate, calcium sulfate, calcium phosphosilicate, sodium phosphate, calcium aluminate, calcium phosphate, hydroxyapatite, tricalcium phosphate, dicalcium phosphate, tricalcium phosphate, tetracalcium phosphate, amorphous calcium phosphate, octacalcium phosphate, and silicates. In certain embodiments the particles may comprise a calcium salt such as calcium carbonate, a zirconium salt such as zirconium dioxide, a zinc salt such as zinc oxide, a magnesium salt such as magnesium silicate, a silicon salt such as silicon dioxide or a titanium salt such as titanium oxide or titanium dioxide. A number of biodegradable and non-biodegradable biocompatible polymers are known in the field of polymeric biomaterials, controlled drug release and tissue engineering (see, for example, U. S. Pat. Nos. 6,123,727; 5,804,178; 5,770,417; 5,736,372; 5,716,404 to Vacanti; U. S. Pat. Nos.

[0190] 6,095,148; 5,837,752 to Shastri; U. S. Pat. No. 5,902,599 to Anseth; U. S. Pat. Nos. 5,696,175; 5,514,378; 5,512,600 to Mikos; U. S. Pat. No. 5,399,665 to Barrera; U. S. Pat. No. 5,019,379 to Domb; U. S. Pat. No. 5,010,167 to Ron; U. S. Pat. No. 4,946,929 to d'Amore; and U. S. Pat. Nos.

[0191] 4,806,621; 4,638,045 to Kohn; see also Langer, Acc. Chem. Res. 33:94, 2000; Langer, J.

[0192] Control Release 62:7, 1999; and Uhrich et al., Chem. Rev. 99:3181, 1999; all of which are incorporated herein by reference).

[0193] The scaffold may be composed of inorganic materials. Inorganic materials include, for instance, magnetic materials, conductive materials, and semiconductor materials. In some embodiments, the scaffold is composed of an organic material.

[0194] In some embodiments, the particles are porous. A porous particle can be a particle having one or more channels that extend from its outer surface into the core of the particle. In some embodiments, the channel may extend through the particle such that its ends are both located at the surface of the particle. These channels are typically formed during synthesis of the particle by inclusion followed by removal of a channel forming reagent in the particle.

[0195] The size of the pores may depend upon the size of the particle. In certain embodiments, the pores have a diameter of less than 15 microns, less than 10 microns, less than 7.5 microns, less

[0196] #14760451vlthan 5 microns, less than 2.5 microns, less than 1 micron, less than 0.5 microns, or less than 0.1 microns. The degree of porosity in porous particles may range from greater than 0 to less than 100% of the particle volume. The degree of porosity may be less than 1%, less than 5%, less than 10%, less than 15%, less than 20%, less than 25%, less than 30%, less than 35%, less than 40%, less than 45%, or less than 50%. The degree of porosity can be determined in a number of ways. For example, the degree of porosity can be determined based on the synthesis protocol of the scaffolds (e.g., based on the volume of the aqueous solution or other channel-forming reagent) or by microscopic inspection of the scaffolds post-synthesis.

[0197] The plurality of particles may be homogeneous for one or more parameters or characteristics. A plurality that is homogeneous for a given parameter, in some instances, means that particles within the plurality deviate from each other no more than about + / - 10%, preferably no more than about + / - 5%, and most preferably no more than about + / - 1% of a given quantitative measure of the parameter. As an example, the particles may be homogeneously porous. This means that the degree of porosity within the particles of the plurality differs by not more than + / - 10% of the average porosity. In other instances, a plurality that is homogeneous means that all the particles in the plurality were treated or processed in the same manner, including for example exposure to the same agent regardless of whether every particle ultimately has all the same properties. In still other embodiments, a plurality that is homogeneous means that at least 80%, preferably at least 90%, and more preferably at least 95% of particles are identical for a given parameter.

[0198] The plurality of particles may be heterogeneous for one or more parameters or characteristics. A plurality that is heterogeneous for a given parameter, in some instances, means that particles within the plurality deviate from the average by more than about + / - 10%, including more than about + / - 20%. Heterogeneous particles may differ with respect to a number of parameters including their size or diameter, their shape, their composition, their surface charge, their degradation profile, whether and what type of agent is comprised by the particle, the location of such agent (e.g., on the surface or internally), the number of agents comprised by the particle, etc. The disclosure contemplates separate synthesis of various types of particles which are then combined in any one of a number of pre-determined ratios prior to contact with the sample. As an example, in one embodiment, the particles may be homogeneous with respect to shape (e.g., at least 95% are spherical in shape) but may be heterogeneous with respect to size, degradation profile and / or agent comprised therein.

[0199] Particle size, shape and release kinetics can also be controlled by adjusting the particle formation conditions. For example, particle formation conditions can be optimized to produce

[0200] #14760451vlsmaller or larger particles, or the overall incubation time or incubation temperature can be increased, resulting in particles which have prolonged release kinetics.

[0201] The particles may also be coated with one or more stabilizing substances, which may be particularly useful for long term depoting with parenteral administration or for oral delivery by allowing passage of the particles through the stomach or gut without dissolution. For example, particles intended for oral delivery may be stabilized with a coating of a substance such as mucin, a secretion containing mucopolysaccharides produced by the goblet cells of the intestine, the submaxillary glands, and other mucous glandular cells.

[0202] To enhance delivery the particles may be incorporated, for instance, into liposomes, virosomes, cationic lipids or other lipid based structures. The term “cationic lipid” refers to lipids which carry a net positive charge at physiological pH. Such lipids include, but are not limited to, DODAC, DOTMA, DDAB, DOTAP, DC-Chol and DMRIE. Additionally, a number of commercial preparations of cationic lipids are available. These include, for example, LIPOFECTIN® (commercially available cationic liposomes comprising DOTMA and DOPE, from GIBCO / BRL, Grand Island, N. Y., USA); LIPOFECTAMINE® (commercially available cationic liposomes comprising DOSPA and DOPE, from GIBCO / BRL); and TRANSFECTAM® (commercially available cationic lipids comprising DOGS in ethanol from Promega Corp., Madison, Wis., USA). A variety of methods are available for preparing liposomes e.g., U. S. Pat. Nos. 4,186,183, 4,217,344, 4,235,871, 4,261,975, 4,485,054, 4,501,728, 4,774,085, 4,837,028, 4,946,787; and PCT Publication No. WO 91 / 17424. The particles may also be composed in whole or in part of GRAS components, i.e., ingredients are those that are Generally Regarded As Safe (GRAS) by the US FDA. GRAS components useful as particle material include non- degradable food based particles such as cellulose.

[0203] In some embodiments, the scaffold is a high molecular weight scaffold that comprises a biological macromolecule, a synthetic macromolecule, or a particle. In some embodiments, the biological macromolecule is a protein, lipid, carbohydrate, or a nucleic acid. In some embodiments, the synthetic macromolecule is a synthetic polymer. In some embodiments, the particle is a nanoparticle or a microparticle. In some embodiments, the scaffold has a total molecular weight greater than 40 kDa.

[0204] Optionally the scaffold may include a biological agent. In one embodiment, a biological agent could be incorporated in the scaffold or it may make up the scaffold. Thus, the compositions of the invention can achieve two purposes at the same time, the diagnostic methods and delivery of a therapeutic agent. In some embodiments the biological agent may be an enzyme inhibitor. In that instance the biological agent can inhibit proteolytic activity at a

[0205] #14760451vllocal site and the detectable marker can be used to test the activity of that particular therapeutic at the site of action.

[0206] Substrates

[0207] The enzyme- specific substrate is a portion of the modular structure that is connected to the scaffold. A substrate, as used herein, is the portion of the modular structure that promotes the enzymatic reaction in the subject, causing the release of a detectable marker. The substrate typically comprises an enzyme- sensitive portion (e.g., protease substrate) linked to a detectable marker. In some embodiments, the substrate comprises an enzyme cleavage site.

[0208] In some instances, the substrate is dependent on enzymes that are active in a specific disease state (e.g., infection). For example, infections are associated with a specific set of enzymes. A nanosensor is designed with one or more substrates that match those of the enzymes expressed by the infectious agent, by the subject in response to the infection or by other diseased tissue. Alternatively, the substrate may be associated with enzymes that are ordinarily present but are absent in a particular disease state. In this example, a disease state would be associated with a lack of signal associated with the enzyme, or reduced levels of signal compared to a normal reference.

[0209] An enzyme, as used herein refers to any of numerous proteins produced in living cells that accelerate or catalyze the metabolic processes of an organism. Enzymes act on substrates. The substrate binds to the enzyme at a location called the active site just before the reaction catalyzed by the enzyme takes place. Enzymes include but are not limited to proteases, glycosidases, lipases, heparinases, phosphatases. In some embodiments, the enzyme is present in a lung of a subject.

[0210] In some embodiments, a substrate comprises an amino acid sequence that is cleaved by an enzyme (e.g., an enzyme- specific substrate). In some embodiments, the enzyme- specific substrate comprises an amino acid sequence cleaved by a serine protease, cysteine protease, threonine protease, aspartic protease, glutamic protease, or a metalloprotease. In some embodiments, the enzyme- specific substrate comprises an amino acid sequence cleaved by trypsin, prolyl peptidase, a matrix metalloproteinase (MMP), a serine protease, granzyme B, cathepsin, or furin.

[0211] In some instances, the substrate is dependent on enzymes that are active in a specific disease state, including, e.g., lung disease, infectious disease, inflammation, and cancer. In some instances, the substrate is dependent on enzymes that are active in a specific stage of disease. See, e.g., Tables 1 and 2 and the Examples below.

[0212] #14760451vlTable 1. Non-limiting examples of disease-associated enzymes and substrates.

[0213] Disease Enzyme Substrate Cancer MMP collagens, gelatin, various ECM proteins Cancer MMP-2 type IV collagen and gelatin

[0214] Cancer MMP-9 type IV and V collagens and gelatin Cancer Kallikreins kininogens,

[0215] plasminogen Cancer Cathepsins broad spectrum of substrates Cancer plasminogen activator, tPA Plasminogen Cancer Urokinase-type plasminogen Plasminogen activator, uPA

[0216] Cancer ADAM (A Diseintegrin And various extracellular Metalloprotease, also MDC, domains of Adamalysin) transmembrane proteins Pancreatic carcinoma MMP-7 various, e.g. collagen 18, FasL, HLE, DCN, IGFBP-3, MAG, plasminogen, other MMPs Pancreatic Cancer ADAM9, ADAM 15 various extracellular domains of transmembrane proteins Prostate adenocarcinoma Matriptase, a type II unspecific, cleaves transmembrane serine protease after Lys or Arg

[0217] residues

[0218]

[0219] #14760451vlDisease Enzyme Substrate Prostate cancer Kallikrein 3 kininogens,

[0220] plasminogen Prostate cancer ADAM 15 various extracellular domains of transmembrane proteins Ovarian carcinoma Kallikrein 6 kininogens,

[0221] plasminogen Epithelial-derived tumors Matriptase, a type II unspecific, cleaves (breast, prostate, ovarian, colon, transmembrane serine protease after Lys or Arg oral) residues Ovarian Cancer MMP-2, MMP-9, kallikrein- 10 type IV and V

[0222] (hk-10) collagens and gelatin, kininogens, plasminogen Breast, gastric, prostate cancer cathepsins B, L and D broad spectrum of substrates Endometrial cancer cathepsin B broad spectrum of substrates esophageal adenocarcinoma cathepsin B broad spectrum of substrates Invasive cancers, metastases type II integral serine proteases

[0223] (dipeptidyl peptidase IV (DPP4 / CD26),seprase / fibroblast

[0224] activation protein alpha

[0225] (FAPalpha) and related type II

[0226] transmembrane prolyl serine

[0227] peptidases))

[0228] Invasive cancers, metastases Seprase various ECM proteins Viral Infections

[0229] All Retroviruses viral protease precursor GagPol fusion

[0230]

[0231] #14760451vlDisease Enzyme Substrate

[0232] HIV HIV protease (HIV PR, an precursor Gag and aspartic protease) GagPol proteins Hepatitis C NS3 serine protease viral precursor polyprotein Dengue Dengue protease autocleavage (NS2B / NS3), NS3 / NS4A and NS4B / NS5 cleavage West Nile NS2B / NS3pro viral precursor polyprotein Bacterial Infections

[0233] Legionella spp. zinc metalloprotease Me-Arg-Pro-Tyr Meninogencephalitis histolytic cysteine protease

[0234] Streptococcus pyogenes (Group streptococcal pyrogenic exotoxin extracellular matrix, A Streptococcus) B (SpeB) immunoglobulins, complement components Clostridium difficile Cwp84 fibronectin, laminin, vitronectin and other ECM proteins Pseudomonas aeruginosa lasA Leu-Gly-Gly-Gly-Ala (SEQ ID NO: 1) Pseudomonas aeruginosa Large ExoProtease A Cleavage of peptide ligands on PARI, PAR2, PAR4 (Protease- activated receptor). See, e.g., Kida et al, Cell Microbiol. 2008 Jul;10(7):1491-504. Pseudomonas aeruginosa protease IV complement factors, fibrinogen, plasminogen (See, e.g., Engel et al., J Biol Chem. 1998 Jul 3;273(27): 16792-7). Pseudomonas aeruginosa alkaline protease Complement factor

[0235]

[0236] C2 (See, e.g., Laarman #14760451vlDisease Enzyme Substrate

[0237] et al., J Immunol. 2012 Jan l;188(l):386-93). Additional Diseases

[0238] Alzheimer’s disease B ACE- 1,2 (Alzheimer secretase) P-amyloid precursor protein

[0239] Stroke and recovery MMP, tPA

[0240] cardiovascular disease Angiotensin Converting Enzyme angiotensin I,

[0241] (ACE) bradykinin Atherosclerosis cathepsin K, L, S broad spectrum of substrates

[0242] arthritis MMP-1 triple-helical fibrillar collagens rheumatoid arthritis thrombin Osteopontin Malaria SUB1 KITAQDDEES (SEQ ID NO: 2) osteoarthritis thrombin Osteopontin osteoporosis / osteoarthritis cathepsin K, S broad spectrum of substrates Arthritis, inflammatory joint Aggrecanase (ADAMTS4, aggrecans

[0243] disease AD AMTS 11) (proteoglycans) thrombosis factor Xa (thrombokinase) Prothrombin thrombosis ADAMTS13 von Willebrand factor (vWF)

[0244] thrombosis plasminogen activator, tPA Plasminogen

[0245] Stress-induced Renal pressure Prostasin epithelial Na channel natriuresis subunits

[0246]

[0247] Table 2. Non-limiting examples of substrates associated with disease and other conditions. DISEASE TARGET SUBSTRATE ENZYME

[0248] Inflammation Interleukin 1 beta MMP-2, MMP-3, MMP-9,

[0249] Trypsin, chymotrypsin, pepsin, Lys-C, Glu-C, Asp-N, Arg-C Pituitary gland IGFBP-3 MMP-1, MMP-3, MMP-9, dysfunction, abnormal Trypsin, chymotrypsin, pepsin,

[0250]

[0251] Lys-C, Glu-C, Asp-N, Arg-C #14760451vlDISEASE TARGET SUBSTRATE ENZYME

[0252] bone density, growth

[0253] disorders

[0254] Cancer TGF-beta MMP-9, Trypsin, chymotrypsin, pepsin, Lys-C, Glu-C, Asp-N, Arg-C

[0255] Cancer, autoimmune TNF MMP-7, Trypsin, chymotrypsin, disease pepsin, Lys-C, Glu-C, Asp-N,

[0256] Arg-C

[0257] Cancer, autoimmune FASL MMP-7, Trypsin, chymotrypsin, disease pepsin, Lys-C, Glu-C, Asp-N,

[0258] Arg-C

[0259] Wound healing, cardiac HB-EGF MMP-3, Trypsin, chymotrypsin, disease pepsin, Lys-C, Glu-C, Asp-N,

[0260] Arg-C

[0261] Pfeiffer syndrome FGFR1 MMP-2, Trypsin, chymotrypsin, pepsin, Lys-C, Glu-C, Asp-N, Arg-C

[0262] Cancer Decorin MMP-2, MMP-3, MMP-7, Trypsin, chymotrypsin, pepsin, Lys-C, Glu-C, Asp-N, Arg-C Cancer Tumor associated Endoglycosidases carbohydrate antigens

[0263] Cancer Sialyl LewisaO-glycanase

[0264] Cancer Sialyl LewisxO-glycanase

[0265] Cancer / Rheumatoid VEGF Trypsin, chymotrypsin, pepsin, Arthritis, pulmonary Lys-C, Glu-C, Asp-N, Arg-C hypertension

[0266] Cancer EGF Trypsin, chymotrypsin, pepsin,

[0267] Lys-C, Glu-C, Asp-N, Arg-C Cancer IL2 Trypsin, chymotrypsin, pepsin,

[0268] Lys-C, Glu-C, Asp-N, Arg-C Cancer IL6 Trypsin, chymotrypsin, pepsin, inflammation / angiogenesis Lys-C, Glu-C, Asp-N, Arg-C Cancer IFN-y Trypsin, chymotrypsin, pepsin,

[0269] Lys-C, Glu-C, Asp-N, Arg-C Cancer TNF-a Trypsin, chymotrypsin, pepsin, inflammation / angiogenesis, Lys-C, Glu-C, Asp-N, Arg-C Rheumatoid Arthritis

[0270] Cancer, Pulmonary TGF-P Trypsin, chymotrypsin, pepsin, fibrosis, Asthma Lys-C, Glu-C, Asp-N, Arg-C Cancer, Pulmonary PDGF Trypsin, chymotrypsin, pepsin, hypertension Lys-C, Glu-C, Asp-N, Arg-C Cancer, pulmonary Fibroblast growth factor Trypsin, chymotrypsin, pepsin, cystadenoma (FGF) Lys-C, Glu-C, Asp-N, Arg-C Cancer Brain-derived neurotrophic Trypsin, chymotrypsin, pepsin, factor (BDNF) Lys-C, Glu-C, Asp-N, Arg-C Cancer Interferon regulatory Trypsin, chymotrypsin, pepsin,

[0271]

[0272] factors (IRF-1, IRF-2) Lys-C, Glu-C, Asp-N, Arg-C

[0273] #14760451vlDISEASE TARGET SUBSTRATE ENZYME

[0274] Inhibitor of tumor MIF Trypsin, chymotrypsin, pepsin, suppressors Lys-C, Glu-C, Asp-N, Arg-C Lymphomas / carcinomas, GM-CSF Trypsin, chymotrypsin, pepsin, alveolar proteinosis Lys-C, Glu-C, Asp-N, Arg-C Cancer invasion M-CSF Trypsin, chymotrypsin, pepsin, Lys-C, Glu-C, Asp-N, Arg-C Chemical carcinogenesis, IL- 12 Trypsin, chymotrypsin, pepsin, multiple sclerosis, Lys-C, Glu-C, Asp-N, Arg-C rheumatoid arthritis,

[0275] Crohn’s disease

[0276] Natural Killer T cell IL- 15 Trypsin, chymotrypsin, pepsin, leukemias, inflammatory Lys-C, Glu-C, Asp-N, Arg-C bowel disease, rheumatoid

[0277] arthritis

[0278] Cirrhosis Tissue inhibitor of MMPs Trypsin, chymotrypsin, pepsin, (TIMPs) Lys-C, Glu-C, Asp-N, Arg-C Cirrhosis Collagen I, III MMP-1, MMP-8, Trypsin,

[0279] chymotrypsin, pepsin, Lys-C, Glu-C, Asp-N, Arg-C Cirrhosis Collagen IV, V MMP-2, Trypsin, chymotrypsin, pepsin, Lys-C, Glu-C, Asp-N,

[0280]

[0281] Arg-C

[0282] For instance, a particular lung disease may be associated with a specific set of enzymes and the specific set of enzymes may distinguish one lung disease from another.

[0283] Lung diseases include but are not limited to lung cancer, interstitial lung disease (ILD), and chronic obstructive pulmonary disease (COPD), and lung infections. The lung diseases may be primary or secondary diseases.

[0284] There are at least two types of lung cancer (e.g., non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC)). NSCLC accounts for about 85% of lung cancer cases and include adenocarcinoma, squamous cell carcinoma and large cell carcinoma. NSCLC may be characterized into stages I- IV by assessing the size and extent of the primary tumor, whether or not the cancer has spread to nearby lymph nodes and metastasis to distant sites (e.g., brain bones, kidneys, liver, or adrenal glands, or other lung). See, e.g., American Joint Committee on Cancer. Lung. In: AJCC Cancer Staging Manual. 8th ed. New York, NY: Springer; 2017: 431-456. SCLC includes small cell carcinoma (oat cell cancer) and combined small cell carcinoma.

[0285] In some embodiments, the lung cancer is EML4-ALK-positive lung cancer.

[0286] Interstitial lung disease (ILD) refers to disorders that cause fibrosis of the lungs. Nonlimiting examples of ILDs include sarcoidosis, asbestosis, hypersensitivity pneumonitis, and idiopathic pulmonary fibrosis. In some cases, ILD is caused by exposure to hazardous chemicals, medical treatments, or medications.

[0287] #14760451vlChronic obstructive pulmonary disease (COPD) may also be referred to as chronic bronchitis or emphysema. COPD is often characterized by obstructed airflow and difficulty breathing. Causes of COPD include tobacco smoke, air pollution and genetic alterations (e.g., alterations resulting in alpha 1 antitrypsin deficiency).

[0288] Infections or infectious diseases are diseases associated with an infectious agent (e.g., pathogens including bacteria, viruses, fungi, and protozoa). Non-limiting examples of pathogenic bacteria include Streptococcus pneumoniae, Staphylococcus aureus, Streptococcus pygenes, Haemophilus influenza, Klebsiella pneumoniae, escherichia coli, Pseudomonas aeruginosa, Mycoplasma pneumoniae, Legionella spp, Anaerobic bacteria, Mycobacterium tuberculosis, Mycoplasma spp, Coxiella burnelil, Chlamydia psittaci, Chlamydia trachomatis, and Chylamydia pneumoniae. Non-limiting examples of viral pathogens include adenoviruses, influenza viruses, and respiratory syncytial viruses. Infections caused by pathogens include pneumonia and bronchitis. In some embodiments, an infection (e.g., an infection- specific) protease is an infectious agent-derived protease that is not present in a host (e.g., an infectious agent- specific protease). In some embodiments, an infection- specific protease is a protease that is not in healthy subjects or in samples from healthy subjects. In some embodiments, an infection- specific protease is a protease that is present in one type of infection but not in another type of infection. In some embodiments, an infection is a lung infection.

[0289] In some embodiments, an infection is associated with a virulence factor (e.g., a protease secreted by an infectious agent). In some embodiments, an infectious agent- specific (e.g., Pseudomonas aeruginosa-specific) protease is LasA (e.g., UniProtKB - Q02L18), Large ExoProtease A (LepA, e.g., UniProtKB - Q02L18), protease IV (e.g., UniProtKB - P08395), Protease IV, or alkaline protease (AprA, e.g., UniProtKB - Q4Z8K9). A non-limiting example of a LasA substrate is a sequence comprising the amino acid sequence LGGGA (SEQ ID NO: 1).

[0290] In some embodiments, an infection is associated with a host factor (e.g., a protease secreted by an immune cells). For example, neutrophil elastase (ELA, e.g., NP_001963.1) is often secreted by neutrophils in response to an infection. A non-limiting example of a neutrophil elastase substrate includes AAFA (SEQ ID NO: 3) and Nle(O-Bzl)-Met(O)2-Oic-Abu. See, e.g., Kasperkiewicz, P. PNAS. 2014; 11(7): 2518-2523).

[0291] In some instances, a protease substrate comprises unnatural amino acids. Unnatural amino acids include 6-benzyloxynorleucine (Nle(O-Bzl)), methionine dioxide (Met(O)2), octahydroindolecarboxylic acid (OIC); and a- aminobutyric acid (Abu).

[0292] #14760451vlA nanosensor may be designed with one or more substrates that match those of the enzymes expressed by diseased tissue (e.g., lung disease tissue). Alternatively, the substrate may be associated with enzymes that are ordinarily present but are absent in a particular disease state. In this example, a disease state would be associated with a lack of signal associated with the enzyme, or reduced levels of signal compared to a normal reference.

[0293] An enzyme, as used herein refers to any of numerous proteins produced in living cells that accelerate or catalyze the metabolic processes of an organism. Enzymes act on substrates and may be derived from a host or an infectious agent (e.g., pathogen associated with an infection). The substrate binds to the enzyme at a location called the active site just before the reaction catalyzed by the enzyme takes place. Enzymes include but are not limited to proteases, glycosidases, lipases, heparinases, phosphatases.

[0294] In some embodiments, a substrate comprises an amino acid sequence that is cleaved by an enzyme (e.g., a protease substrate). In some embodiments, the enzyme-specific substrate comprises an amino acid sequence cleaved by a serine protease, an alkaline protease, a lysinespecific protease, cysteine protease, threonine protease, aspartic protease (e.g., Asp A), glutamic protease, and / or a metalloproteinase (i.e.: metalloprotease). As their names suggest, serine, cysteine, threonine, and aspartic proteases use a catalytic serine, cysteine, threonine, or aspartate residue, respectively, for catalysis. Mechanistically, metalloproteinases use a metal in catalysis. In some embodiments, the protease is trypsin, prolyl peptidase, a granzyme, cathepsin, or furin. In some embodiments, the protease is granzyme B. In some embodiments, the target protease is an endopeptidase. In some embodiments, the target protease is an exopeptidase.

[0295] As used herein, a substrate (e.g., protease substrate) may be enzymatically cleaved by one or more proteases (e.g., at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 30, at least 40, at least 50, at least 60, or at least 100) proteases.

[0296] A nanosensor of the present disclosure may detect the activity of an endogenous and / or an exogenous protease. An endogenous protease is a protease that is naturally produced by a subject (e.g., subject with a particular disease or a host with an infection). An exogenous protease is a protease that is not naturally produced by a subject and may be produced by an infectious agent (e.g., a bacteria, a fungi, protozoa, or a virus). In some embodiments, a protease is only expressed by a subject (e.g., a human) and not by an infectious agent. In some embodiments, a protease is infectious agent- specific and is only produced by an infectious agent not by the infectious agent’s host. Without being bound by a particular theory, a nanosensor that

[0297] #14760451vlcomprises a substrate for an infectious agent- specific protease would not be cleaved by a hostspecific protease. In some embodiments, an infectious agent- specific protease is produced by one infectious agent but not another. Such infectious agent- specific proteases may be useful in distinguishing between different infectious agent-induced diseases. In some embodiments, a protease that is produced by a host, an infectious agent or both, but is not active does not promote the release of a detectable marker from a nanosensor.

[0298] A substrate may be attached directly to the scaffold. For instance it may be coated directly on the surface of microparticles using known techniques, or chemically bonded to a polymeric scaffold, such as a PEG scaffold (e.g., via a peptide bond). Additionally, the substrate may be connected to the scaffold through the use of a linker. In some embodiments the scaffold has a linker attached to an external surface, which can be used to link the substrate. Another molecule can also be attached to the linker. In some embodiments, two molecules are linked using a transpeptidase, for example Sortase A.

[0299] The substrate is preferably a polymer made up of a plurality of chemical units. A “chemical unit” as used herein is a building block or monomer which may be linked directly or indirectly to other building blocks or monomers to form a polymer (e.g., a multi-arm PEG scaffold).

[0300] Non-limiting examples of nanosensors and compositions comprising nanosensors

[0301] A nanosensor described herein may comprise any of the scaffolds, enzyme substrates, enzyme cleavage sites, and / or dipeptide linkers described herein. In some embodiments, a nanosensor comprises a scaffold linked to an enzyme cleavage site and the enzyme cleavage site is linked to a volatile reporter via a dipeptide linker. In some embodiments, the enzyme cleavage site is present in an enzyme-specific substrate.

[0302] In some embodiments, the enzyme cleavage site is a protease cleavage site. In some embodiments a protease cleavage site is cleaved by a protease. For example, the protease may be a serine protease, cysteine protease, threonine protease, aspartic protease, glutamic protease, or a metalloprotease. In some embodiments, the protease is trypsin, prolyl peptidase, a granzyme, cathepsin, or furin. In some embodiments, the protease is granzyme B. In some embodiments, the protease is an endopeptidase. In some embodiments, the protease is an exopeptidase.

[0303] In some embodiments, the nanosensor comprises a volatile reporter selected from 2,2,3,3,3-pentafluoropropylamine (HFA1), 1H,1H-perfluoropentylamine (HFA3), methanol, ethanol, 2-propanol, 2-butanol, ethanol- 2- propanol -d?, and 2-butanol-&. In some

[0304] #14760451vlembodiments, the nanosensor comprises a volatile reporter selected from methanol, ethanol, 2-propanol, 2-butanol, ethanol-?, 2-propanol-oO, and 2-butanol-oO.

[0305] In some embodiments, a nanosensor described herein comprises a sequence set forth in any one of Tables 3A-3C. In some embodiments, a nanosensor described herein comprises a protease cleavage site set forth in any one of Tables 3A-3C. As a non-limiting example, for S108-Ome in Table 3A, the protease cleavage site is between arginine and glycine (before the dipeptide linker). In some embodiments, a nanosensor described herein comprises a volatile organic compound set forth in any one of Tables 3A-3C.

[0306] In some embodiments, a nanosensor described herein comprises a sequence selected from the group consisting of: GAANLTRGp (SEQ ID NO: 5); GGPGp (SEQ ID NO: 6);

[0307] GKPLGLp (SEQ ID NO: 7); GGILSRIp (SEQ ID NO: 8); GPLGMRGp (SEQ ID NO: 9);

[0308] GGPFGCHAKGp (SEQ ID NO: 11); GGAIEFD (SEQ ID NO: 12); GGPLGL (SEQ ID NO: 13); GGPVPLp (SEQ ID NO: 14); GRQRRSp (SEQ ID NO: 15); GGP; and GGPFGCHAK (SEQ ID NO: 16), in which a lower case letter indicates a d- amino acid.

[0309] A composition may comprise any of the nanosensors described herein. In some embodiments, a composition described herein comprises one or more nanosensors (e.g., 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more 10 or more, 11 or more, 12, or more, 13 or more, 14 or more, 15 or more, or 20 or more nanosensors). In some embodiments, a composition described herein comprises a nanosensor with an endopeptidase cleavage site. In some embodiments, a composition described herein comprises a nanosensor with an exopeptidase cleavage site. In some embodiments, a composition described herein comprises a nanosensor with an endopeptidase cleavage site and a nanosensor with an exopeptidase cleavage site. In some embodiments, the one or more nanosensors each comprise a sequence set forth in any one of Tables 3A-3C. In some embodiments, a composition described herein comprises one or more nanosensors (2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more 10 or more, 11 or more, or 12 nanosensors) each comprising an amino acid sequence selected from the group consisting of: GAANLTRGp (SEQ ID NO: 5); GGPGp (SEQ ID NO: 6); GKPLGLp (SEQ ID NO: 7); GGILSRIp (SEQ ID NO: 8);

[0310] GPLGMRGp (SEQ ID NO: 9); GGPFGCHAKGp (SEQ ID NO: 11); GGAIEFD (SEQ ID NO: 12); GGPLGL (SEQ ID NO: 13); GGPVPLp (SEQ ID NO: 14); GRQRRSp (SEQ ID NO: 15); GGP; and GGPFGCHAK (SEQ ID NO: 16), in which a lower case letter indicates a d-amino acid.

[0311] In some embodiments, a composition described herein consists of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nanosensors. In some embodiments, each

[0312] #14760451vlnanosensor comprises a sequence set forth in any one of Tables 3A-3C. In some embodiments, a composition described herein consists of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 nanosensors each comprising an amino acid sequence selected from the group consisting of: GAANLTRGp (SEQ ID NO: 5); GGPGp (SEQ ID NO: 6); GKPLGLp (SEQ ID NO: 7); GGILSRIp (SEQ ID NO: 8); GPLGMRGp (SEQ ID NO: 9); GGPFGCHAKGp (SEQ ID NO: 11); GGAIEFD (SEQ ID NO: 12); GGPLGL (SEQ ID NO: 13); GGPVPLp (SEQ ID NO: 14); GRQRRSp (SEQ ID NO: 15); GGP; or GGPFGCHAK (SEQ ID NO: 16), in which a lower case letter indicates a d-amino acid.

[0313] In some embodiments, a composition consists of the nanosensors comprising the substrate sequences set forth in Table 3A. In some embodiments, a composition consists of the nanosensors comprising the substrate sequences set forth in Table 3B. In some embodiments, a composition consists of the nanosensors comprising the substrate sequences set forth in Table 3C.

[0314] In some embodiments, a composition comprises two or more (e.g., three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, eleven or more, twelve or more, thirteen or more, fourteen or more, or fifteen or more) of the nanosensors described herein. In some embodiments, a composition comprises:

[0315] a first nanosensor comprising a scaffold linked to an enzyme cleavage site, wherein the enzyme cleavage site is linked to a volatile reporter via a dipeptide linker; and

[0316] a second nanosensor comprising a second scaffold linked to a second enzyme cleavage site, wherein a second volatile reporter is directly attached to the second enzyme cleavage site, optionally wherein:

[0317] the second scaffold is linked to the second enzyme cleavage site via a peptide, and the second volatile reporter is attached at a C-terminus of the peptide.

[0318] In some embodiments, the first nanosensor comprises an endopeptidase cleavage site. In some embodiments, the second nanosensor comprises an exopeptidase cleavage site. In some embodiments, the first nanosensor comprises an endopeptidase cleavage site, and the second nanosensor comprises an exopeptidase cleavage site. In some embodiments, the first nanosensor comprises a volatile reporter selected from methanol, ethanol, 2-propanol, 2-butanol, ethanol- s, 2-propanol-rf?, and 2-butanol-rfj. In some embodiments, the second volatile reporter is 2, 2, 3,3,3-pentafluoropropylamine (HFA1) or 1H,1H-perfluoropentylamine (HFA3). In some embodiments, the volatile reporter is selected from methanol, ethanol, 2-propanol, 2-butanol, ethanol-iA, 2-propanol-?, and 2-butanol-t, and the second volatile reporter is 2, 2, 3,3,3-pentafluoropropylamine (HFA1) or 1H,1H-perfluoropentylamine (HFA3). In some embodiments, the first nanosensor comprises an amino acid sequence selected from the group #14760451vlconsisting of: GAANLTRGp (SEQ ID NO: 5); GGPGp (SEQ ID NO: 6); GKPLGLp (SEQ ID NO: 7); GGILSRIp (SEQ ID NO: 8); GPLGMRGp (SEQ ID NO: 9); GGPFGCHAKGp (SEQ ID NO: 11); GGPVPLp (SEQ ID NO: 14); GRQRRSp (SEQ ID NO: 15); and GGP, in which a lower case letter indicates a d-amino acid. In some embodiments, the second nanosensor comprises a second amino acid sequence selected from the group consisting of GGAIEFD (SEQ ID NO: 12); GGPLGL (SEQ ID NO: 13); and GGPFGCHAK (SEQ ID NO: 16). In some embodiments, the first nanosensor comprises an amino acid sequence selected from the group consisting of: GAANLTRGp (SEQ ID NO: 5); GGPGp (SEQ ID NO: 6); GKPLGLp (SEQ ID NO: 7); GGILSRIp (SEQ ID NO: 8); GPLGMRGp (SEQ ID NO: 9); GGPFGCHAKGp (SEQ ID NO: 11); GGPVPLp (SEQ ID NO: 14); GRQRRSp (SEQ ID NO: 15); and GGP, in which a lower case letter indicates a d-amino acid; and the second nanosensor comprises a second amino acid sequence selected from the group consisting of GGAIEFD (SEQ ID NO: 12); GGPLGL; (SEQ ID NO: 13) and GGPFGCHAK (SEQ ID NO: 16).

[0319] In some embodiments, a composition comprises two or more nanosensors disclosed herein and the composition comprises two or more enzyme cleavage sites (e.g., at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 25, at least 30, at least 40, or at least 50 enzyme cleavage sites). In some embodiments, the two or more enzyme cleavage sites are on different nanosensors. In some embodiments, at least two enzyme cleavage sites (e.g., at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 25, at least 30, at least 40, or at least 50 enzyme cleavage sites) are cleaved by the same protease. In some embodiments, at least two enzyme cleavage sites (e.g., at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 25, at least 30, at least 40, or at least 50 enzyme cleavage sites) are different. In some embodiments, at least two enzyme cleavage sites (e.g., at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 25, at least 30, at least 40, or at least 50 enzyme cleavage sites) are cleaved by different proteases. In some embodiments, at least two enzyme cleavage sites (e.g., at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 25, at least 30, at least 40, or at least 50 enzyme cleavage sites) are different but are cleaved by the same protease. In some

[0320] #14760451vlembodiments, at least two enzyme cleavage sites (e.g., at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 25, at least 30, at least 40, or at least 50 enzyme cleavage sites) are different and are cleaved by different proteases.

[0321] Methods of detecting enzyme activity and detecting disease

[0322] Compositions (e.g., nanosensors) described herein can be administered to any suitable subject. As used herein, a subject is a human, non-human primate, cow, horse, pig, sheep, goat, dog, cat, or rodent. The subject may have, be at risk for, or is suspected of having a disease (e.g., an infectious disease, cancer, inflammation and / or a lung disease). In some embodiments, the subject has relapse of a cancer. In some embodiments, the subject has previously been treated for a disease.

[0323] A nanosensor described herein may be used to monitor the effect of a particular treatment. A nanosensor may be administered more than once (e.g., at least twice, at least three times, at least four times, at least five times, or at least 10 times) to a subject. In some embodiments, the subject has been treated with a therapeutic agent (e.g., a chemotherapy, an antibiotic, a targeted agent including a various kinase inhibitors). A subject may be treated with a therapeutic agent prior to the first administration of a nanosensor or be treated with a therapeutic agent after administration of a nanosensor (e.g., between two administrations of a nanosensor, or after one administration of a nanosensor).

[0324] The enzyme nanosensors of the disclosure are administered to the subject in an effective amount for detecting enzyme activity. An “effective amount”, for instance, is an amount necessary or sufficient to cause release of a detectable level of volatile reporter in the presence of an enzyme. The effective amount of a composition described herein may vary depending upon the specific composition used, the mode of delivery of the composition, and whether it is used alone or in combination with other compounds (e.g., a composition comprising a multiplexed library of nanosensors or combined with administration of a therapeutic agent). The effective amount for any particular application can also vary depending on such factors as the disease being assessed or treated, the particular compound being administered, the size of the subject, or the severity of the disease or condition as well as the detection method. One of ordinary skill in the art can empirically determine the effective amount of a particular molecule of the invention without necessitating undue experimentation. Combined with the teachings provided herein, by choosing among the various active compounds and weighing factors such as

[0325] #14760451vlpotency, relative bioavailability, patient body weight, severity of adverse side-effects and preferred mode of administration, an effective regimen can be planned.

[0326] Pharmaceutical compositions of the disclosure comprise an effective amount of one or more agents, dissolved or dispersed in a pharmaceutically acceptable carrier. The phrases “pharmaceutical or pharmacologically acceptable” refers to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to an animal, such as, for example, a human, as appropriate. Moreover, for animal (e.g., human) administration, it will be understood that preparations should meet sterility, pyrogenicity, general safety and purity standards as required by FDA Office of Biological Standards.

[0327] As used herein, “pharmaceutically acceptable carrier” includes any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, drugs, drug stabilizers, gels, binders, excipients, disintegration agents, lubricants, sweetening agents, flavoring agents, dyes, such like materials and combinations thereof, as would be known to one of ordinary skill in the art (see, for example, Remington's Pharmaceutical Sciences (1990), incorporated herein by reference). Except insofar as any conventional carrier is incompatible with the active ingredient, its use in the therapeutic or pharmaceutical compositions is contemplated. The agent may comprise different types of carriers depending on whether it is to be administered in solid, liquid or aerosol form, and whether it need to be sterile for such routes of administration as injection.

[0328] Any suitable route of administration may be used. In some instances, the nanosensor is administered through inhalation. In some instances, the nanosensor is administered to the pulmonary space. In some instances, the nanosensor is administered to a lung of a subject. In some embodiments, the nanosensor is administered intratracheally. In some instances, the nanosensor is administered intravenously, intranasally, subcutaneously, or any combination thereof. See also, e.g., WO2019173332A1.

[0329] Any suitable method known in the art or disclosed herein may be used to detect a volatile reporter that has been released from the nanosensor. As a non-limiting example, a biological sample (e.g., breath sample, blood sample, feces sample, urine sample, sputum sample, sweat sample) may be collected from a subject who has been administered a nanosensor of the present disclosure and the biological sample may be assayed to detect a released volatile reporter. In some instances, the biological sample is a blood culture, a sputum culture, or a combination thereof. In some instances, the level of a released volatile reporter in a sample obtained from a subject who has been administered a nanosensor is compared relative to the level of the

[0330] #14760451vlendogenous levels of the released volatile reporter. In some instances, an increase in the presence of the volatile reporter relative to the level of the volatile reporter from a healthy subject is indicative of the subject having a disease. In some instances, the presence of a released synthetic volatile reporter in a biological sample obtained from a subject who has been administered a nanosensor is indicative of the subject having a disease.

[0331] In some embodiments, a nanosensor described herein is used to classify a stage of disease (e.g., local early-stage disease, advanced disease, metastatic disease, or relapsed disease) local, early-stage lung cancer in mice. Clinically, nanosensors may be an effective alternative to screening by low-dose CT, reducing cost, exposure to harmful radiation and, in theory, false positive rates. Nanosensors described herein may also have utility in serially monitoring patients for progression, remission and recurrence, or even molecularly profiling tumors in vivo.

[0332] In some embodiments, a method described herein comprises one or more steps selected from:

[0333] (a) administering a nanosensor of the present disclosure to a subject;

[0334] (b) detecting in a breath sample obtained from a subject that has been administered a nanosensor of the present disclosure one or more volatile reporters that have been released from one or more nanosensors when exposed to an enzyme present in the subject;

[0335] (c) classifying a subject that has been administered a nanosensor of the present disclosure as having lung cancer; and

[0336] (d) classifying a subject that has been administered a nanosensor of the present disclosure as having a relapse of a lung cancer, optionally wherein the subject had previously received treatment for the lung cancer.

[0337] In some embodiments, a method described herein comprises detecting in a sample (e.g., breath sample) obtained from a subject who has been administered a nanosensor described herein: one or more volatile reporters that have been released from the nanosensor when the nanosensor was exposed to an enzyme present in the subject. In some embodiments, the subject has been administered alectinib, a first-line ALK tyrosine kinase inhibitor (TKI) for advanced ALK+non-small cell lung cancers (NSCLCs). See, e.g., Wang et al., J. Thorac. Dis. 15, 1935— 1947 (2023). In some embodiments, a nanosensor described herein is administered or has been administered to a subject who has been treated with one or more doses of a tyrosine kinase inhibitor.

[0338] In some embodiments, a method disclosed herein comprises administering a composition consisting essentially of five nanosensors to a subject.

[0339] #14760451vlIn some embodiments, a composition disclosed herein is used to detect activity of an enzyme and / or the presence of a disease. In some embodiments, a composition disclosed herein is used to detect relapse of a disease. In some embodiments, the composition is administered to a subject. In some embodiments, the composition comprises two or more enzyme cleavage sites (e.g., at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 25, at least 30, at least 40, or at least 50 enzyme cleavage sites). In some embodiments, at least two enzyme cleavage sites (e.g., at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 25, at least 30, at least 40, or at least 50 enzyme cleavage sites) are cleaved by the same enzyme. In some embodiments, the cleavage sites are different cleavage sites. In some embodiments, at least two enzyme cleavage sites (e.g., at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 25, at least 30, at least 40, or at least 50 enzyme cleavage sites) are cleaved by different enzymes.

[0340] In some embodiments, a composition disclosed herein is used to a subject to detect activity of more than one enzyme (e.g., at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 25, at least 30, at least 40, or at least 50 enzymes). In some embodiments, the composition comprises two or more enzyme cleavage sites (e.g., at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 25, at least 30, at least 40, or at least 50 enzyme cleavage sites). In some embodiments, at least two enzyme cleavage sites (e.g., at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 25, at least 30, at least 40, or at least 50 enzyme cleavage sites) are cleaved by different proteases.

[0341] Any of the methods disclosed herein, may further comprise detecting one or more endogenous VOCs. In some embodiments, a method disclosed herein comprises detecting an exogenous VOC (e.g., an exogenous VOC that is released from a nanosensor disclosed herein) and an endogenous VOC. As a non-limiting example, machine learning analysis of synthetic vABN signals together with endogenous VOCs may be used to detect a disease in a subject. In some embodiments, an endogenous VOC is hexanal, which may be useful for detecting lung

[0342] #14760451vlcancer. In some embodiments, a method disclosed herein further comprises detecting and / or analyzing circulating tumor (ctDNA), protein biomarkers, and / or circulating tumor cells.

[0343] Without wishing to be being bound by a particular theory, multimodal diagnostics combining detection of an exogenous VOC released from a nanosensor disclosed herein and detection and / or analysis of an endogenous VOC, ctDNA, protein biomarkers and / or circulating tumor cells may enhance diagnostic accuracy (e.g., detection of a disease) over one modality. In some embodiments, miniature and / or high resolution devices may be used in the detection of an exogenous VOC released from a nanosensor disclosed herein, which may be useful for point-of-care diagnostics. In some embodiments, a miniature and / or high resolution device includes 3D compact mass spectrometers and / or optical spectrometers. In some embodiments, the optical spectrometers are portable optical spectrometers. In some embodiments, the optical spectrometers are portable Raman spectrometers. In some embodiments, a method disclosed herein may be useful in early detection of disease, better patient stratification and improved outcomes in protease-mediated diseases.

[0344] EXAMPLES

[0345] The ability of a protease to cleave any given substrate depends on the chemical and spatial arrangements of substrate amino acids relative to the cleavage site (P1 / P1').33For example, significant reduction in the cleavage kinetics of a trypsin-sensing probe, S10834, was observed upon substitution of glycine at Pl’ to phenylalanine (FIG. 6). It was sought to generate a volatile-releasing mechanism that preserves cleavage site specificity, in order to detect various types of endopeptidase activities. To this end, proteolytic cleavage was leveraged to liberate dipeptide fragments that subsequently underwent intramolecular aminolysis, a nucleophilic reaction between proximate primary amine and ester groups to generate diketopiperazine and an alcohol. Proline-containing dipeptides are enthalpically favored toward the desired products. It was determined whether protease-cleavable probes where proline at P2’ of a substrate was conjugated to an alkyl ester at P3’ would allow for selectivity at P1 / P1' following release of an alcohol reporter by aminolysis (FIG. 2A)

[0346] To investigate the effect of P1' residue on the kinetics of alcohol release by aminolysis, a panel of acid-sensitive tert-butyloxycarbonyl (boc)-protected dipeptide methyl esters (Ome), or Xaa-Pro-Ome, were synthesized, where Xaa is glycine (G), alanine (A), serine (S), leucine (L), or phenylalanine (F) in the order of increasing amino acid side chains. These amino acids are also found among the most frequently observed Pl’ residues in the MEROPS protease database40. The boc group was removed by trifluoroacetic acid, after which methanol generated

[0347] #14760451vlin the headspace was analyzed by mass spectrometry, following aminolysis of the deprotected dipeptides in phosphate buffered saline at pH 7 at 37 °C (FIG. 2B and FIGs. 7A-7C). As shown in FIG. 2C, deprotected GP-Ome exhibits rapid generation of methanol and plateaus within 20 min, suggesting equilibrium in the gas phase. The relative ranking of methanol release rates is in the order of G> A> S> L> F, such that the rate decreases with increasing size of the side chains (i.e., amount of time needed to reach rate=0; FIGs. 2D-2E). Regardless of the size of Pl’ residue, reactions were completed within 1 hour. Notably, non-specific hydrolysis at the ester group was absent in common buffers at physiological pH of 5.5, 7, and 9, except when using a high concentration (50 mM) of amine-containing tris buffer (FIG. 8). These observations suggest that using an aminolysis reaction to achieve volatile release is compatible for a diagnostic test.

[0348] The effects of P2’ were further investigated by substituting proline with its analogs (FIG.

[0349] 2F), which are frequently used to modulate protease substrate specificity. The findings herein show that rapid methanol release, particularly from dipeptides containing 4- or 5-member rings at P2’, but not from those with side chains on the pyrrolidine rings (FIG. 2G). The energy difference between cis-trans conformers of each dipeptide was also investigated using density functional theory (DFT) calculations, which suggested that efficient aminolysis is facilitated by a preference for cis conformation (FIG. 2H).

[0350] The volatile sensing activity-based nanosensor (vABN) is composed of an eight-arm polyethylene glycol (PEG) nanoscaffold attached to a protease substrate and exogenous reporter (FIG. 3A). To examine alcohol release following selective protease cleavage, a trypsin-cleavable substrate, S10834, was modified with methyl ester. The resulting modification, designated S108-Ome (Ac-GAANLTRGP-Ome) (SEQ ID NO: 26), yields a substrate that can be canonically cleaved by human Trypsin-3 (PRSS3) after the arginine residue, and result in methanol release by aminolysis. The molecular weights of any intact peptide and cleaved fragments present in the liquid phase were monitored by MALDI-TOF mass spectrometry (FIG. 3B), while volatile methanol generated by aminolysis of fragment GP-Ome was detected in the headspace (FIG. 3C). To demonstrate that alcohol release is dependent on cleavage site specificity, S108-Ome with kallikrein-14 (FIG. 3C, bottom) was incubated, but in this reaction no methanol was detected (FIG. 3D), due to this enzyme utilizing a different cleavage site between Pl asparagine and Pl’ threonine (FIG. 3B). The selectivity towards target proteases could be further enhanced by substituting 1-proline with d-proline at P2’, which prevented non-specific release of alcohols by prolyl peptidases, like fibroblast activation protein (FAP), which is involved in inflammation and cancer42(FIGs. 3E-3F). To prevent off-target cleavage, the d-proline at P2’ was used in all

[0351] #14760451vlsubsequent vABNs in this work. Further, methanol release was well correlated with PRSS3 activity, which is known to be active at neutral and basic pH but not in acidic conditions (FIG.

[0352] 3G).

[0353] Trypsins, such as PRSS3, are known to be involved in the host entry and replication of influenza A virus.29,30To demonstrate that S108 activity is specific to trypsin-like proteases, S108 ethyl-d5 ester, which can release biocompatible ethanol-d5, was screened against PRSS3, the closely -related human airway trypsin-like protease (HAT), and 14 other non-trypsin recombinant human proteases, and only observed meaningful alcohol release in response to trypsin activity (FIG. 3H). To test the ability of S108 vABNs to detect infection- driven trypsin activity an Influenza A (A / Puerto Rico / 8 / 34, PR8) mouse model was generated according to a method used in previous studies28(FIG. 4A). Longitudinal samples of bronchoalveolar lavage fluids (BALF), which acts as a proxy for the lung airspace, were collected from PR8-infected mice. Over the course of infection, elevated trypsin levels were detected by enzyme-linked immunosorbent assays (ELISAs) and increasing trypsin activity using S108-Ome in BALF (FIGs. 4B-4C). It was then sought to test whether these BALF signals correlated to breath by intratracheally delivering S108 vABNs to healthy and PR8-infected mice on day 6 postinfection. Breath collected 10 min post-vABN delivery showed increased levels of ethanol-d5 in infected mice versus healthy controls (FIG. 4D), suggesting that the S108 vABN could be used to non-invasively detect trypsin activity that is associated with viral infection.

[0354] Having established a volatile release mechanism that can generate breath-based disease signal, it was next sought to test the feasibility of multiplexing vABNs in breath. A panel of five vABNs was constructed, each comprising a different peptide substrate-reporter pair. Two of the vABNs were synthesized with the reporters pentafluoropropylamine (HFA1) and perfluoroamine (HFA3), which have terminal amines that are compatible with the previous vABN chemistry for sensing exopeptidase-like activity. The other three vABNs comprised alcohol reporters, including ethanol-d5, 2-propanol-d7, and 2-butanol-d3 for endopeptidases. Four substrate sequences (S8, S108, S72 and S26, Table 3B) were selected and were modified to increase susceptibility to influenza-associated proteolytic host responses. This panel also included S70, also known as BV01, which is susceptible to cleavage by granzyme B activity and was recently identified as a highly sensitive probe for distinguishing host responses in viral versus bacterial pneumonia. The 5-plex vABN cocktail was delivered directly into the lungs of PR8-infected mice, and breath was collected 10 mins later. Multiplexed signals were standardized via z-score and subjected to machine learning analysis using a random forest classifier (FIGs. 4E-4F). Diagnostic accuracy was measured by the receiver operating

[0355] #14760451vlcharacteristic (ROC) curve, which distinguished infected mice from healthy controls with an area under the curve (AUC) of 0.95 and an out-of-bag error rate of 2.14%, suggesting high accuracy without significant overfitting (FIG. 4G). Performance of the trained classifier was also visualized with a confusion matrix (FIG. 4H). The AUC values from individual vABNs suggested that multiplexing enhanced overall accuracy, as the combined diagnostic power outperformed individual sensors alone (FIG. 41).

[0356] Tables 3A-3C. Sequences of vABNs.

[0357] Note: lowercase indicates d-amino acids. 2,2,3,3,3-pentafluoropropylamine (CAS: 422-03-7), 1H,1H-perfluoropentylamine (CAS: 355-27-1), ethanol-d5 (CAS: 1859-08-1), 2-propanol-d7 (CAS: 19214-96-7), 2-butanol-d3 (CAS: 53716-61-3). Ac: acetyl group, Cy5: cyanine 5 fluorophore, and PEG: polyethylene glycol.

[0358] Table 3A. All substrates for in vitro headspace assessments.

[0359] Name Substrate Cleaved Dipeptide Volatile Target Figure Sequence fragment linker protease

[0360] sequence

[0361] S108- Ac- Ac- GP Methanol Trypsin FIGs. 3B- Ome GAANLTRGP- GAANLTR 3D, 3G,

[0362] Ome (SEQ ID (SEQ ID NO: 4C NO: 26) 17)

[0363] S108(D- Ac- Ac- Gp Methanol Trypsin FIGs. 3E-pro)- GAANLTRGp- GAANLTR 3F Ome Ome (SEQ ID (SEQ ID NO:

[0364] NO: 27) 17)

[0365] S108- Ac- Ac- Gp Ethanol-d5 Trypsin FIG. 3H Od5eth GAANLTRGp- GAANLTR

[0366] Od5eth (SEQ ID (SEQ ID NO:

[0367] NO: 28) 17)

[0368] PP01- Ac-GGPGp-Ome Ac-GGP Gp Methanol Prolyl FIG. 9A Ome (SEQ ID NO: 29) peptidase

[0369] PP09- Ac-GKPLGLp- Ac-GKPLG Lp Methanol Metalloprotease FIGs. 9A- Ome Ome (SEQ ID NO: 9G (SEQ ID NO: 30) 18)

[0370] PP10- Ac-GGILSRIp- Ac-GGILSR Ip Methanol Serine protease FIG. 9A Ome Ome (SEQ ID NO:

[0371] (SEQ ID NO: 32) 19)

[0372] PP12- Ac-GPLGMRGp- Ac-GPLGMR Gp Methanol Serine protease FIG. 9A Ome Ome (SEQ ID NO:

[0373] (SEQ ID NO: 34) 20)

[0374] PP13- Ac- Ac- Gp Methanol Serine protease FIG. 9A Ome GGPFGCHAKGp- GGPFGCHAK

[0375] Ome (SEQ ID NO:

[0376]

[0377] (SEQ ID NO: 36) 16)

[0378] #14760451vlTable 3B. All substrates for in vivo influenza A in FIGs. 4D-4I.

[0379] vABN Substrate Sequence Cleaved Dipeptide Volatile Target protease fragment linker

[0380] sequence

[0381] S70 Ac-CKK(Cy5)-PEG4- Ac- — 2, 2, 3,3,3- Granzyme B GGAIEFD-HFA1 CKK(Cy5)- Pentafluoropropylamine

[0382] (SEQ ID NO: 37) PEG4- GGAIEFD

[0383] S8 Ac-CKK(Cy5)-PEG4- Ac- 1H,1H- CathepsinsGGPLGL-HFA3 CKK(Cy5)- Perfluoropentylamine

[0384] (SEQ ID NO: 41) PEG4- GGPLGL

[0385] (SEQ ID

[0386] NO: 21)

[0387] S108 Ac-CKK(Cy5)-PEG4- Ac- Gp Ethanol-d5 TrypsinGAANLTRGp- CKK(Cy5)- Od5eth (SEQ ID NO: PEG4- 28) GAANLTR

[0388] (SEQ ID

[0389] NO: 17)

[0390] S72 Ac-CKK(Cy5)-PEG4- Ac- Lp 2-propanol-d7 Metalloproteases GGPVPLp- CKK(Cy5)- Od7isoprop (SEQ ID PEG4- NO: 38) GGPVP

[0391] (SEQ ID

[0392] NO: 22)

[0393] S26 Ac-CKK(Cy5)-PEG4- Ac- Sp 2-butanol-d3 Furin GRQRRSp-Od3but CKK(Cy5)- (SEQ ID NO: 39) PEG4- GRQRR

[0394] (SEQ ID

[0395]

[0396] NO: 23)

[0397] Table 3C. All substrates for in vivo Eml4-Alk in FIGs. 5A-5I.

[0398] vABN Substrate Cleaved Dipeptide Volatile Target Sequence fragment linker protease sequence

[0399] PP01 Ac-CKK(Cy5)- Ac- — 2, 2, 3,3,3- Prolyl PEG4-GGP- CKK(Cy5)- Pentafluoropropylamine peptidase HFA1 PEG4-GGP

[0400] PP13 Ac-CKK(Cy5)- Ac- 1H,1H- Serine protease PEG4- CKK(Cy5)- Perfluoropentylamine GGPFGCHAK- PEG4- HFA3 (SEQ ID GGPFGCHAK

[0401] NO: 40) (SEQ ID NO:

[0402] 16)

[0403] PP10 Ac-CKK(Cy5)- Ac- Gp Ethanol-d5 Serine protease

[0404]

[0405] PEG4- CKK(Cy5)- #14760451vlvABN Substrate Cleaved Dipeptide Volatile Target Sequence fragment linker protease sequence

[0406] GPLGMRGp- PEG4- Od5eth (SEQ GPLGMR

[0407] ID NO: 35) (SEQ ID NO:

[0408] 20)

[0409] PP12 Ac-CKK(Cy5)- Ac- Ip 2-propanol-d7 Serine protease PEG4- CKK(Cy5)- GGILSRIp- PEG4- Od7isop (SEQ GGILSR (SEQ

[0410] ID NO: 33) ID NO: 19)

[0411] PP09 Ac-CKK(Cy5)- Ac- Lp 2-butanol-d3 Metalloprotease PEG4- CKK(Cy5)- GKPLGLp- PEG4- Od3but (SEQ GKPLG (SEQ

[0412]

[0413] ID NO: 31) ID NO: 18)

[0414] Having established that 5 distinct VOC reporters that can be analyzed collectively to accurately detect active influenza infection, the multiplexing approach was then applied to the detection of lung cancer, the global leading cause of cancer mortality.31'43,44

[0415] A subset of the urinary ABNs (PP01, 9, 10, 12, and 13) were re-engineered into a new 5-plex vABN panel. First, to validate that the modified substrates can release volatile reporters following proteolytic cleavage, each sequence was modified with methyl ester, incubated in BALF collected from healthy and Eml4-Alk mice at 7 weeks post tumor initiation, and methanol concentration was measured in the reaction headspace. Differential signals were detected between the healthy and tumor-bearing mice, particularly for sensors PP09 and PP10, which were designed to detect activity of MMPs and serine proteases, respectively (FIG. 9A and Table 3C). After confirming that these substrates were capable of volatile release, they were synthesized into multiplexed vABNs. These five vABNs use the same volatile reporters as the PR85-plex, but contain entirely different substrate sequences (Table 3C). Micro-computed tomography (microCT) scans were obtained prior to all breath collections. Multiplexed vABNs were delivered to the lungs of healthy and Eml4-Alk mice at 4-, 5-, or 6-weeks after tumor initiation (FIG. 5 A). Breath was collected 10, 30, and 60 min after vABN delivery and volatile signal amplitudes were integrated over 30 min by area under the curve analysis (AUC, FIGs. 10A-10E). The multiplexed signals generated within 30 min post- vABN delivery were standardized and subjected to machine learning analysis using the gradient boosting algorithms46. Diagnostic accuracies were measured by ROC curves, which distinguished Eml4-Alk mice from healthy controls with combined AUCs of 0.64 at 4 weeks, 0.79 at 5 weeks, and 0.93 at 6 weeks (FIG. 5B). Five-fold cross validations were performed to evaluate the classifiers, #14760451vlof which mean AUCs of the folds and Fl and precision scores were reported in Table 4. The AUC values of individual vABNs suggested that multiplexing enhanced the overall accuracy (FIG. 5C) of the diagnostic.

[0416] Table 4. Cross validation scores for the evaluation of classifiers in FIGs. 5A-5I. Five-fold cross validations were performed on the classifiers using Python (v3.9.0) and the scikit-learn446(v 1.6) package.

[0417] Dataset Mean AUC of folds Fl score

[0418] 4- week Eml4-Alk (FIG. 5B) 0.61 + 0.35 0.68 + 0.22 5-week Eml4-Alk (FIG. 5B) 0.83 + 0.05 0.74 + 0.12 6-week Eml4-Alk (FIG. 5B) 0.71 + 0.3 0.72 + 0.16

[0419]

[0420] 10- week relapse (FIG. 5BH) 0.70 + 0.19 0.76 + 0.08

[0421] In addition to monitoring for the growth of lung tumors, multiplexed vABNs detected altered protease activity patterns in mice following treatment with alectinib (FIGs. 5D-5E). Principle component analysis (PCA) revealed that protease activity in vehicle-treated Eml4-Alk mice diverged from healthy controls, whereas that of alectinib-treated mice were more similar to healthy controls (FIG. 5F). The MMP specificity of PP09 was also explored by incubating methanol probe PP09-Ome with recombinant MMP9, MMP12, and MMP13, which are elevated in the transcriptomic data of Eml4-Alk mice24. As shown in FIGs. 9B-9G, elevated methanol signal was observed specifically for MMP9, which agreed with the elevated protein levels in BALF quantified by ELISAs.

[0422] The effective regression of tumors by alectinib treatment was observed by microCT scans (FIG. 5G, top). In clinical trials, high initial response rates to tyrosine kinase inhibitors have been observed for this patient population; however, many patients acquired resistance to TKIs and ultimately relapse on therapy within 1 to 2 years.47,48It was therefore sought to assess this approach in detecting tumor relapse. To this end, alectinib treatment was terminated after two weeks. As indicated by microCT scans, mice treated with alectinib that previously bore tumors relapse three weeks after discontinuation of the drug (FIG. 5G, bottom). Similar to the approach above, multiplexed vABNs were delivered to healthy and relapsed mice and the standardized volatile signatures were used to train classifiers. As shown in FIG. 5H, these results showed high accuracy of classification, offering a feasible method to monitor ALK relapse. Notably, elevated signals were observed for multiple vABNs between treatment and relapse in the same cohort, revealing altered pulmonary protease activity in relapsing tumors (FIG. 51).

[0423] Discussion

[0424] #14760451vlPresented herein is the design and credential of a chemistry-based approach to multiplex breath-based synthetic biomarkers by coupling disease-associated protease activity with distinct exogenous volatile reporters. Multiplexed volatile activity-based nanosensors (vABNs), when combined with analysis by machine learning algorithms, accurately classified disease states in preclinical models of viral infection and lung cancer using rapid breath tests. Importantly, this approach was extended to monitor both treatment response and relapse in a mouse model of Alk-mutant lung cancer, highlighting the modular nature of vABNs, and their suitability for adaptation to various pathophysiological processes that currently require monitoring by invasive procedures.

[0425] The vABN disclosed herein comprises a nanoscaffold attached to protease- sensing substrate and exogenous reporter. To achieve multiplexing in breath, a method of detecting dysregulated protease activities of broad catalytic types is needed. Effective mechanisms for sensing endopeptidase activity, which accounts for >50% of known 589 human proteases40'50,51have remained out of reach. In some embodiments, the present disclosure addresses these limitations by incorporating a reaction to release volatile reporters upon selective endopeptidase cleavage, including several disease-associated metallo- and serine proteases. Without being bound by a particular theory, the mechanism couples protease scission with conformational-constrained motifs to facilitate rapid head-to-tail cyclization of proline-containing dipeptide fragments via aminolysis, along with alcohol production. In some aspects, the instant specification describes the use of intramolecular aminolysis as a volatile release mechanism for breath readouts. Dipeptides comprising Pl’ amino acids — glycine, alanine, serine, and leucine -complete reactions within 30 min, allowing for rapid breath tests. Proline analogs, including d-proline, azetidine, and homoproline, were also identified that promoted the m-configurations required for intramolecular aminolysis, thereby expanding the scope of detectable protease activities in vivo.

[0426] This platform was first applied to an influenza mouse model by designing a 5-plex panel that was capable of detecting protease activities associated with viral infection. Each vABN was attached to either an amine- or alcohol-based reporter that was distinguishable by mass and did not exist in the breath. This multiplexed panel included several probes, highlighting S108 that was demonstrated to be cleaved specifically by trypsin in this work, and S70 (or BV01). These vABNs alone were able to classify infected mice from healthy controls with AUC values of -0.90, with an improved performance when multiplexed (AUC of 0.95). This platform was then extended to an / \ / A- mutant lung cancer model by generating a unique set of 5-plex vABN panel adapted from previous urinary ABNs that differentiated tumor bearing mice from healthy

[0427] #14760451vlcontrols. Analysis of multiplexed signals by machine learning algorithms provided accurate assessments of tumor progression and treatment response 30 min post administration with enhanced classification performances compared to single vABNs. These results demonstrate modularity of the platform disclosed herein to diagnose complex disease with nuanced signature via breath.

[0428] Among the five vABNs, volatile signals from PP09, PP10, and PP12 vABNs exhibited an overall increase in the vehicle-treated mice and regression with alectinib treatment. Notably, the responses of PP09 and PP10 agreed with previous urinary diagnostics of lung cancers24,25but not PPI 2. Without being bound by a particular theory, PPI 2, now with a cleavage site at arginine for serine proteases, may be cleaved by different proteases (e.g., MMP) in the urinary panel, which could be confirmed - for example, by leveraging inhibitors to selectively abrogate protease activities. Overall, the MMP-specific PP09 was the most sensitive vABN for monitoring tumor progression and treatment-related changes. As an endopeptidase, the volatilereleasing chemistry allowed for identifying cleavage sensitivity and site specificity to PP09 by subclasses of tumor- associated MMPs. These findings demonstrate a high modularity and the complementary nature of ABN and vABN platforms when used for multimodal readout in urine and breath. This new vABN platform was further applied to monitoring the recurrence of ALK+NSCLCs, where resistance to first-line ALK inhibitors often leads to clinical relapse within one to two years.47,48Clinical resistance to TKIs can occur through a variety of mechanisms. As a proof-of-concept, alectinib treatment of mice was discontinued, and functional relapse of this model was scored based on tumor regrowth, and which was universally observed within three weeks after drug withdrawal. The multiplexed vABNs detected changes in protease activity signatures associated with relapse in mice that had previously experienced tumor regression under alectinib treatment, supporting the clinical potential of this approach to monitor disease state and ongoing treatment efficacy.

[0429] These findings highlight the promise of combining synthetic biomarkers with breath analysis for future point-of-care diagnostics. The multiplexed breath test may decode disease much sooner than a urinary readout that typically takes around 2 hrs for reporters to be voided into the urine. To further increase disease specificity, future efforts could focus on extending the multiplexing capacity to include additional disease-relevant proteases. Synthetic vABN signals can also be analyzed together with subsets of standardized, disease-associated endogenous VOCs11to increase diagnostic accuracy and disease specificity. Further, additional steps to optimize vABN formulations for human use are required before translating this approach to clinical settings. Encouragingly, chemistry capable of releasing ethanol-, a biocompatible

[0430] #14760451vlreporter being evaluated in a phase 2 trial (EVOLUTION), shows promise for translation and integration with non-protease markers (e.g., EVOC® probe52for p -glucuronidase) to develop multi-analyte breath tests. In some embodiments, combining orthogonal diagnostic approaches that leverage ctDNA, protein biomarkers, and / or circulating tumor cells may also enhance diagnostic accuracy over any one modality alone. Finally, integrating this technology with portable breath analysis devices (e.g., ReCIVA® breath sampler), could facilitate its implementation in point-of-care diagnostics, which may contribute to earlier detection, better patient stratification, and improved outcomes in protease-mediated diseases.

[0431] Methods

[0432] Peptide synthesis. / -Butyloxycarbonyl (boc) protected dipeptide alkyl esters and protease substrate alkyl esters were synthesized by CPC Scientific or the Biopolymers and Proteomics Core at the Swanson Biotechnology Center at the Koch Institute of MIT. Briefly, peptides were synthesized by standard solid phase synthesis. To couple desired alcohol reporters to the peptides, Steglich esterification53was first used to prepare boc- Yaa- alkyl ester, where Yaa is proline or its derivatives. Boc was removed by anhydrous trifluoroacetic acid (TFA) followed by coupling to the remaining peptide fragment. All protecting groups were removed in final TFA treatment. Peptide products were purified by high performance liquid chromatography (HPLC) and analyzed by matrix-assisted laser desorption / ionization time-of-flight (MALDI-TOF) mass spectrometry.

[0433] Density Functional Theory (DFT) calculations. The energy of dipeptide conformers was calculated using ORCA (v5.0). The initial geometry was generated by ChemDraw (20.1.1, Revvity Signals) and optimized using the built-in Universal Force Field in Avogadro (vl.2.0). DFT geometry optimization was then performed with the B3LYP functional. The def2-TZVP basis set, which provides triple-zeta quality and polarization functions for greater accuracy in representing electron distributions, was applied. The RIJCOSX approximation, supported by the def2 / J auxiliary basis set, was used to speed up the calculation of the Coulomb and exchange integrals through a resolution of the identity (RI) approach. The KDIIS algorithm was applied to enable efficient convergence of the self-consistent field (SCF) procedure. Finally, Grimme's D3B J dispersion correction was included to account for van der Waals interactions, which are often underestimated in DFT calculations.

[0434] In vitro headspace analysis. Aminolysis reaction in FIGs. 2A-2H was performed as follows. Boc-protected dipeptide methyl esters were dissolved in dichloromethane (DCM) and aliquoted to ~10 pmol in 1.7 mL tubes. After evaporating DCM, anhydrous 30% trifluoracetic

[0435] #14760451vlacid (TFA) in DCM was incubated with the peptides for ~3 hrs at room temperature to deprotect the boc group. After evaporation of TFA and DCM, 100 pL of phosphate buffer (50 mM) was added to the deprotected dipeptides and adjusted to pH~7 with 10 M sodium hydroxide. In FIGs.

[0436] 3A-3H, protease cleavage mediated aminolysis was performed by incubating peptide alkyl esters (100 pM) with target proteases (100 nM, R& D Systems or Enzo Life Sciences) in a total volume of 100 pL for 1 hr at 37 °C. The released alcohols in the headspace were analyzed by proton transfer reaction mass spectrometry (PTR-MS, lonicon).

[0437] Animal models. All animal studies were approved by the MIT Institutional Animal Care and Use Committee (protocols 2301000462 and 2203000310) and were conducted in compliance with institutional and national policies. Influenza A A / PR / 8 / 34 (PR8) model was generated by dosing 7-week-old female mice (BALB / c, Taconic) with 25 or 30 pL of Influenza virus through intranasal instillation. Eml4-Alk lung cancer model was generated in 6-week-old female mice (C57BL / 6J, Jackson Labs) by intratracheal instillation of 50 pL adenovirus expressing the Ad-EA vector (VQAd Cas9 ALK EML4072415; Viraquest; 1.5*108PFU in Opti-MEM with 10 mM CaCh)32. These mice are referred to as “Eml4-Alk” mice. The criteria for euthanasia, as dictated by the MIT Committee on Animal Care, was body weight loss of greater than 20% for PR8 mice and 10% for Eml- Aik mice, significant dyspnea, or poor body condition. Animals were monitored daily for PR8 mice and weekly for Eml4-Alk mice throughout studies, and the criteria for euthanasia were not met. Healthy control cohorts were age- and sex-matched mice that did not undergo instillation of influenza virus or adenovirus.

[0438] In vivo characterization of vABNs. Peptides for the nanosensors were synthesized by CPC Scientific. The peptides containing an N-terminal cysteine were covalently conjugated to 8-arm 40 kDa PEG maleimide with a tripentaerythritol core (JenKem Technology) at a molecular ratio of 2:1 in water overnight at room temperature. The vABNs were prepared in phosphate buffered saline and delivered to the lungs by intratracheal instillation (50 pL total volume). Breath collection method was reported previously23. In brief, at 10-, 30-, and 60-mins post vABN delivery, mice were placed in 10 mL syringes connected to closed Luer lock stopcock valves for 2 min. Next, 25-gauge needles were connected to the apparatus and inserted through the rubber septum of 12 mL evacuated Exetainers® (Labco). Headspace (10-12 mL) was transferred from the syringe into the Exetainer® and measured by PTR-MS.

[0439] Alectinib treatment of Eml4-Alk mice. Eml4-Alk mice were randomized to receive either control drug vehicle or alectinib (MedChemExpress), at 20 mg / kg prepared directly in drug vehicle, daily by oral gavage for 14 consecutive days. The drug vehicle comprises 0% (v / v) dimethylsulfoxide (DMSO; Sigma Aldrich), 10% (v / v) Cremophor EL (Sigma Aldrich), 15%

[0440] #14760451vl(v / v) polyethylene glycol)-400 (PEG400; Sigma Aldrich), and 15% (w / v) (2-Hydroxypropyl)-P-cyclodextrin (Sigma Aldrich). Mice were monitored daily. Investigators were not blind with respect to treatment.

[0441] Machine learning analysis of multiplexed vABN data. Selection of vABN substrates was performed using analytic pipelines of the Protease Activity Analysis (PAA) package34. Analyses of reporter data were performed using Python (v3.9.0) and the scikit-leam54(vl.6) package. For all in vivo vABN experiments, z-score standardization was performed on volatile reporter signals in the same mice cohort prior to statistical analysis. Differential vABN signals were subjected to unpaired two-tailed t tests. Multiple comparisons were analyzed by one-way or two-way ANOVAs followed by correction for multiple hypotheses using the Bonferroni method. Padj < 0.05 was considered significant.

[0442] In FIGs. 4G-4I, machine learning classification was performed based on the vABN signatures of datasets comprising paired features (z-scored vABN signals) and labels (the class membership; for example, PR8 or healthy). The classification was performed using a random forest classifier with 100 trees. Estimates of out-of-bag error were used for cross-validation, and trained classifiers were tested on the independent test cohort.

[0443] In the Eml4-Alk model, principal component analysis (PCA, FIG. 5F) and machine learning classification (FIGs. 5B, 5C, 5H, and 51) were performed based on the vABN signatures of datasets comprising paired features (the z-scored vABN signals) and labels (Eml4-Alk or healthy). In FIGs. 5B, 5C, 5H, and 51, classification of vABN signatures was performed by 25:75 test-train splits of the datasets and trained on gradient boosting classifiers.

[0444] Hyperparameters for the classifiers were determined by GridSearchCV from the scikit-leam46,54package. A five-fold cross validation was performed on the classifiers, where mean AUC and the Fl score were reported (Table 4). Performances of the classifiers were evaluated with the area under the curves (AUCs) of the receiver operating curves (ROC).

[0445] Statistics and reproducibility

[0446] PCA and machine learning classifications of vABN data were performed in Python (v.3.9.0) and the scikit-learn54(vl.6) package. All remaining statistical analyses were conducted in Prism 9.0 (GraphPad). Sample sizes, statistical tests, and P-values are specified in Brief Descriptions of the Drawings. All in vitro and in vivo vABN experiments were repeated at least twice with similar results.

[0447] Code availability

[0448] #14760451vlCode for substrate selections was published as part of the Protease Activity Analysis (PAA) package28. The codes for analyzing multiplex data in FIGs. 4G-4I and FIGs. 5B, 5C, 5F, 5H, 51 are available at github.com / christinewang76 / multiplex-mass-analysis.git.

[0449] REFERENCES

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[0509] #14760451vl

Claims

CLAIMSWhat is claimed is:

1. A nanosensor comprising a scaffold linked to an enzyme cleavage site, wherein the enzyme cleavage site is linked to a volatile reporter via a dipeptide linker.

2. The nanosensor of claim 1, wherein the enzyme cleavage site is a protease cleavage site.

3. The nanosensor of claim 2, wherein the protease cleavage site is cleaved by a protease, optionally wherein the protease is selected from trypsin, prolyl peptidase, a matrix metalloproteinase (MMP), a serine protease, granzyme B, cathepsin, and furin.

4. The nanosensor of any preceding claim, wherein the dipeptide linker comprises the amino acid sequence -Xaa-Xp-, wherein:Xaa is selected from glycine (Gly), alanine (Ala), serine (Ser), leucine (Leu), isoleucine (He), and phenylalanine (Phe); andXp is selected from proline (Pro), D-proline (D-pro), and a proline replacement.

5. The nanosensor of claim 4, wherein the proline replacement is of formula:

6. The nanosensor of claim 4 or 5, wherein Xp is Pro, D-Pro, or a proline replacement,wherein the proline replacement is of formula:or a D-isomer thereof.

7. The nanosensor of any preceding claim, wherein the volatile reporter is a perfluorocarbon or an alcohol, or an isotopically labeled derivative thereof, optionally wherein the isotopically labeled derivative of the alcohol is a deuterated alcohol.#14760451vl8. The nanosensor of claim 7, wherein the perfluorocarbon is 2, 2, 3,3,3-pentafluoropropylamine (HFA1) or 1H,1H-perfluoropentylamine (HFA3), the alcohol is methanol, ethanol, 2-propanol, or 2-butanol, or the deuterated alcohol is ethanol-rf, 2-propanol-cb, or 2-butanol-r.

9. A composition comprising:the nanosensor of any preceding claim; anda second nanosensor comprising a second scaffold linked to a second enzyme cleavage site, wherein a second volatile reporter is directly attached to the second enzyme cleavage site, optionally wherein:the second scaffold is linked to the second enzyme cleavage site via a peptide, and the second volatile reporter is attached at a C-terminus of the peptide.

10. A method comprising one or more steps selected from:(a) administering a nanosensor of any one of claims 1-8 or a composition of claim 9 to a subject;(b) detecting in a breath sample obtained from a subject that has been administered a nanosensor of any one of claims 1-8 or a composition of claim 9 one or more volatile reporters that have been released from one or more nanosensors when exposed to an enzyme present in the subject;(c) classifying a subject that has been administered a nanosensor of any one of claims 1-8 or a composition of claim 9 as having lung cancer; and(d) classifying a subject that has been administered a nanosensor of any one of claims 1-8 or a composition of claim 9 as having a relapse of a lung cancer, optionally wherein the subject had previously received treatment for the lung cancer.#14760451vl

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