Mycobacteriophage functionalized magnetic nanocrystal clusters for highly sensitive detection of tuberculosis and non-tuberculosis mycobacterium (NTM) related infection

Abstract

A rapid and cost-effective bacilli enrichment technology is provided that combines magnetic nanotechnologies with bacteriophage. The bacteriophage provides recognition functionality, while the magnetic nanocrystal clusters have excellent separation efficiency. Using portable and inexpensive devices, one can achieve rapid, point-of-care detection of tuberculosis, Non-Tuberculosis Mycobacterium (NTM) and urinary tract infection (UTI) in clinically relevant matrices, including sputum, urine, and blood, with a detection limit of 500 cfu / mL for BCG within 35 min. Excellent potential for clinical tuberculosis diagnostics in resource-limited settings is demonstrated.

Description

[0001] MYCOBACTERIOPHAGE FUNCTIONALIZED

[0002] MAGNETIC NANOCRYSTAL CLUSTERS FOR HIGHLY

[0003] SENSITIVE DETECTION OF TUBERCULOSIS AND NON¬

[0004] TUBERCULOSIS MYCOBACTERIUM (NTM) RELATED INFECTION

[0005] FIELD OF THE INVENTION

[0006] This invention relates to the detection of Mycobacterium tuberculosis and nontuberculosis Mycobacterium (NTM) using magnetic nanotechnology and bacteriophage. In particular, the invention relates to the conjugation of bacteriophage with magnetic nanocrystal clusters as the separation agent for tuberculosis and non-tuberculosis bacteria in complicated systems (such as patient sputum samples). The target bacteria can be efficiently captured by a handheld magnet using magnetic separation. The magnetically enriched bacteria is then read out with the ATP reagent using a portable luminometer.

[0007] BACKGROUND OF THE INVENTION

[0008] Mycobacterium tuberculosis (Mtb), responsible for tuberculosis, is one of the most dangerous and infectious pathogens due to its rapid spread and acute death toll. Upon inhalation of the bacilli-containing droplets from other active tuberculosis patients, Mtb cells could invade the host lung and eventually enter different areas, such as the brain, kidney, lymphatic system, and bloodstream.

[0009] The World Health Organization estimated that 10.6 million people were affected with tuberculosis in 2022, and 1.6 million died due to tuberculosis- related illness. Most infections and deaths occur in developing countries in Africa and South Asia. It is crucial to achieve an accurate clinical diagnosis of Mtb in both tuberculosis active patients and latent, asymptomatic carriers to prevent the spread and facilitate the cure for tuberculosis. Nevertheless, a rapid and point-of-care detection protocol remains challenging in resource-poor settings.

[0010] Sputum smear microscopy is a simple and inexpensive technique for diagnosing pulmonary tuberculosis. Yet, it suffers from low sensitivities with a limit of detection (LOD) higher than 104colony -forming units per milliliter (cfu / mL). More importantly, fluorescent staining cannot distinguish dead cells from viable ones. The bacterial culture-based protocol is currently a reliable but timeconsuming detection method for Mtb: it can take up to eight weeks due to their prolonged growth on the solid medium; thus, they cannot provide timely diagnosis and prevent transmission. Culture-independent methods, such as nucleic acid-based detection, are now commonly used. However, PCR-based panels are often susceptible to false positives as they do not report bacteria viability, and the high cost makes them less accessible to resource-limited settings. Alternatively, molecular recognition agents highly specific to target bacteria, such as antimicrobial peptides, antibodies, aptamers, and enzymatic activity-based probes, have been prepared as biosensors for various pathogen detection. Although promising, these molecular recognition agents are usually expensive, and only limited numbers of bacterial aptamers or antibodies have been identified. The expense and availability of these biomolecules substantially limit their applicability in biosensor-based detection for clinical use. Therefore, recognition agents with low costs and high performance for Mtb detection are highly desirable, which is the topic of this invention.

[0011] SUMMARY OF THE INVENTION

[0012] Rapid and cost-effective bacilli enrichment technology is provided that combines magnetic nanotechnologies with mycobacteriophage for the specific and sensitive detection of Mycobacterium tuberculosis and non-tuberculosis mycobacterium (NTM). The mycobacteriophage provides recognition functionality, while the magnetic nanocrystal clusters have excellent separation efficiency. The bacteriophage propagated in Msmeg was functionalized with dopamine, which caused it to lose the ability to complete a lytic cycle. Electron microscopy images showed the strong binding of the mycobacteria with the nanoparticles coated with Dopa-Phage. The magnetically enriched bacteria maintained viable cellular structures and were disrupted by ultrasound to facilitate the rapid release of cellular ATP. The number of bacteria was quantified using the ATP assay to generate strong bioluminescent signals. A high specificity toward mycobacteria species and nearly 100% separation efficiency were examined.

[0013] Finally, portable and inexpensive devices were used, including a magnet rack, a small ultrasonic cleaner, and a luminometer, to demonstrate rapid, point-of- care tuberculosis detection in clinically relevant matrices, including sputum, urine, and blood, and with other interfering bacteria. A detection limit of 500 cfu / mL for BCG was achieved with a detection time of 35 min. This technology demonstrates excellent potentials for clinical tuberculosis diagnostics in resource-limited settings.

[0014] Applications of embodiments of the invention can be a test kit which could contain the magnetic separation agent (aqueous MNP -Phage solution) in a test tube, a small handheld magnet, and the ATP reagent. After collecting clinical samples (e.g., sputum or urine) from the patients in a test tube, the separation agent is added and incubated at room temperature. Then, the magnet is placed near the test tube to perform magnetic separation. The ATP reagent is then added to the magnetically separated content, and the test tube is transferred to a portable luminometer for immediate signal reading. The test turnaround time is within an hour, and the patient can be informed immediately. Each luminometer can easily handle thousands of tests in a particular area. Customers are likely to be the NGOs that serve in Africa and South Asia to help people get timely tuberculosis diagnosis and treatment.

[0015] The biosensor presented herein is prepared using magnetic clusters conjugated with mycobacteriophage that can result in rapid and cost-effective tuberculosis detection. Bacteriophages are bacterial viruses commonly present in the environment and can infect the target bacteria with high specificity. Magnetic clusters are a novel magnetic nanomaterial formed by clustering nanocrystals (4~10 nm) into controlled aggregates (20-200 nm). They exhibit superior magnetic separation efficiency that can be performed rapidly using a cheap, handheld permanent magnet. Combining the recognition capability of bacteriophages with magnetic clusters can achieve accurate targeting of mycobacterium tuberculosis and non-tuberculosis mycobacterium.

[0016] In one embodiment the invention can be characterized by a bacteriophage functionalized with magnetic nanocrystal clusters. The bacteriophage functionalized with magnetic nanocrystal clusters contains a bacteriophage with nitro-dopamine molecules conjugated to the bacteriophage. Magnetic nanocrystal clusters are anchored to hydroxyl groups of the nitro-dopamine molecules. In one embodiment, the bacteriophage is Mycobacterium smegmatis phage (ATCC 11759-B1) and E. coli phage. The magnetic nanocrystal clusters range in size from 20 nm to 200 nm. Each of the magnetic nano-crystals is about 65 nm in size. A ratio between the nitro-dopamine molecules to the bacteriophage is defined from 90: 1 to 20: 1. In another example, a ratio between the nitro-dopamine molecules to the bacteriophage is about 60: 1.

[0017] BRIEF DESCRIPTION OF THE DRAWINGS

[0018] FIG. 1 shows according to an exemplary embodiment of the invention transmission electron microscopy (TEM) images of the mycobacteriophages with uranyl acetate staining.

[0019] FIG. 2 shows according to an exemplary embodiment of the invention modification of the bacteriophage with nitro-dopamine with a schematic for the conjugation of dopamine to phage via EDC coupling reagent.

[0020] FIGs. 3A-B show according to exemplary embodiments of the invention modification of the bacteriophage with nitro-dopamine with (FIG. 3A) the UV absorbance spectra of nitro-dopamine (10 pg / mL), bacteriophage (1.5*10npfu / mL), and the dopamine functionalized phage with different Dopa / Phage ratios. (FIG. 3B) A The plaque assays of the dopamine functionalized phages. The absence of plaque formation indicates that the phages lose their lytic abilities after chemical modification.

[0021] FIG. 4 shows according to an exemplary embodiment of the invention transmission electron microscopy images of the magnetic clusters. The average cluster diameter is about 65 nm.

[0022] FIG. 5 shows according to an exemplary embodiment of the invention the dynamic light scattering (DLS) of the as-synthesized MNP and MNP -Phage. The MNP -Phage is colloidally stable in an aqueous solution, and after three days, there is no change in their hydrodynamic sizes. Inset: the zeta potential of MNP and MNP- Phage.

[0023] FIG. 6 shows according to an exemplary embodiment of the invention the MNP -Phage have excellent magnetic separation efficiency, in which a complete magnetic separation can happen within 20 min using a handheld permanent magnet. MNP-Phage can be lyophilized for extended periods of storage and can gain complete colloidal stability after sonication.

[0024] FIGs. 7A-C show according to exemplary embodiments of the invention TEM images of MNP-Phage bound to (FIG. 7A) Msmeg, (FIG. 7B) BCG, and (FIG. 7C) Mtb. 106cfu of bacteria were mixed with 100 pg MNP -Phage and magnetically washed three times before TEM sample preparation.

[0025] FIG. 8 shows according to an exemplary embodiment of the invention fluorescent microscopy images of MNP-Phage bound to Msmeg using Auramine O stanning. 105cfu of Msmeg was magnetically separated using 100 pg MNP-Phage, dispersed in 1 mL Auramine O solution (1 mg / mL) and incubated for 30 min. The content was then magnetically washed 3 times to remove the free dye molecules. For fluorescent imaging, the excitation is 438 nm, and the emission is 505 nm.

[0026] FIG. 9 shows according to an exemplary embodiment of the invention additional TEM images of MNP-Phage bound to Msmeg, BCG, and Mtb. 105cfu (top) and 103cfu (bottom) of bacteria were mixed with 100 pg MNP-phage and magnetically washed 3 times prior to TEM sample preparation.

[0027] FIG. 10 shows according to an exemplary embodiment of the invention TEM images of MNP-Phage bound to NTM. 106cfu of M. avium, M. abscessus, and M. scrofulaceum were mixed with 100 pg MNP-phage and magnetically washed 3 times prior to TEM sample preparation. FIG. 11 shows according to an exemplary embodiment of the invention the process of magnetic separation of BCG using MNP -Phage. Fresh cultures of BCG and MNP- Phage were dispersed in 1 mL PBS buffer. After 20 min of incubation, the vial was placed in a magnet rack until a complete separation was achieved. The magnetic content and the supernatant were separately plated on the 7H10 agar plates to observe the bacteria distribution.

[0028] FIGs. 12A-B show according to exemplary embodiments of the invention (FIG. 12A) time-dependent bioluminescent signals of magnetically separated bacteria. 104cfu of Msmeg, BCG, and Mtb were separated using 100 pg MNP -Phage and eventually dispersed in 100 pL of PBS, followed by adding 10 pL of Cell Titer Gio reagent. (FIG. 12B) Sonication on bacteria samples can significantly increase the bioluminescent signals. In a similar set up, the separated bacteria were placed in a bath sonicator for 15 seconds prior to the addition of the 10 pL Cell Titer Gio reagent.

[0029] FIGs. 13A-C show according to exemplary embodiments of the invention bacteria detection using the ATP assay. (FIG. 13A) The bioluminescent signal versus the total BCG (cfu) added prior to magnetic separation with MNP -Phage. The calibration curve indicates a detection limit of 3.5*102cfu BCG. (FIG. 13B) The BCG separation efficiency versus the amount of MNP -Phage used in magnetic separation. The solid and dashed lines indicate the bioluminescent signal of the magnetically separated content and the supernatant solution, respectively. (FIG. 13C) The bioluminescent signal of different bacteria strains after magnetic separation. 104and 105cfu of M. smegmatis, M. bovis BCG, M. tuberculosis, M. avium, M. abscessus, M. scrofulaceum, C. diphtheriae, E. coli, K. pneumoniae, S. pneumoniae, and S. aureus were mixed with MNP -Phage and underwent magnetic separation. The solid line and the grey bar at the bottom of each graph indicate the signal of the blank sample plus three times the standard deviation.

[0030] FIG. 14 shows according to an exemplary embodiment of the invention TEM images of the as-synthesized magnetic clusters and nanoparticles with diameters of 120 nm, 28 nm, and 12.5 nm, respectively.

[0031] FIG. 15 shows according to an exemplary embodiment of the invention the hydrodynamic sizes of the MNP and MNP -Phage with various MNP physical diameters.

[0032] FIGs. 16A-B show according to exemplary embodiments of the invention optimizing the MNP size and Dopa / Phage ratio for bacteria detection. Different numbers of Msmeg were magnetically separated using 100 ug MNP -phage. After 20 min of incubation, the culture tube was placed on a magnet for 20 min to collect Msmeg for ATP assay detection. (FIG. 16A) Comparison between the 120 nm, 65 nm, and 28 nm clusters and the 12 nm nanoparticles. (FIG. 16B) Comparison of the Dopa-to-Phage ratio.

[0033] FIGs. 17A-B show according to exemplary embodiments of the invention the bioluminescent signal versus the total number of Msmeg (FIG. 17A) and Mtb (FIG. 17B) added prior to magnetic separation with MNP-phage. The calibration curve indicates a detection limit of 5*102cfu for Msmeg and 9*102cfu for Mtb.

[0034] FIGs. 18A-C show according to exemplary embodiments of the invention MNP coated with E. coli phage can be applied to separate E. coli. (FIGs. 18A-B) TEM images of MNP -Ephage bound to E. coli. (FIG. 18C) MNP-phage mixed with E. coli. In these tests, 105cfu of Msmeg, BCG, or E. coli were mixed with 100 ug MNP or MNP-phage and magnetically washed 3 times prior to TEM sample preparation. (FIG. 18C) The bioluminescent signal of the ATP assay versus the total number of E. coli (cfu) added prior to magnetic separation with MNP -Ephage. The Calibration curve indicates a detection limit of 2*102cfu E. coli. FIG. 19 shows according to an exemplary embodiment of the invention TEM images of deactivated BCG bound to MNP -Phage.

[0035] FIG. 20 shows according to an exemplary embodiment of the invention the fluorescent images of the magnetically separated BCG stained with FITC-BA.

[0036] FIGs. 21A-B show according to exemplary embodiments of the invention MNP -Phage for the detection of deactivated BCG viability sensing. (FIG. 21A) The fluorescent signal versus the total number of BCG (cfu). (FIG. 21B) Comparison of the bioluminescent signal and the fluorescent signal of the magnetically separated Msmeg (total number of 104cfu) at different viability levels.

[0037] FIGs. 22A-E show according to exemplary embodiments of the invention the point-of-care detection of tuberculosis. (FIG. 22A) Schematics for the detection flow using portable devices for magnetic separation, sonication, and luminescence measurement, with an overall time of 35 min. (FIG. 22B) Different numbers of BCG dispersed in 1 mL water, artificial urine, whole porcine blood, or artificial sputum, followed by magnetic separation and ATP assay detection. (FIG. 22C) l*103cfu of BCG dispersed in various volumes of water, urine, blood, or sputum. (FIG. 22D) Different numbers of BCG mixed with l*106cfu of interfering bacteria (C. diphtheriae, E. coli, S. pneumoniae, or S. aureus). (FIG. 22E) Different numbers of interfering bacteria mixed with l*103cfu BCG. A blank sample was prepared by adding 10 pL ATP reagent in 100 pL PBS, whose bioluminescent signal was arbitrarily set as 1 to normalize the test samples. The solid line and the grey bar represent the signal of the blank sample plus three times the standard deviation.

[0038] FIG. 23 shows according to an exemplary embodiment of the invention ATP assay without magnetic separation. Different numbers of BCG were dispersed in these solutions (0.1 mL). After sonication, 10 pL Cell Titer Gio reagent was directly added.

[0039] FIG. 24 shows according to an exemplary embodiment of the invention Magnetic separation of bacteria at different pH conditions.

[0040] FIGs. 25A-C show according to exemplary embodiments of the invention the point-of-care detection of Mtb. (FIG. 25A) Different numbers of attenuated Mtb H37Rv dispersed in 1 mL water, artificial urine, whole porcine blood, or artificial sputum, followed by magnetic separation and ATP assay detection. (FIG. 25B) 1 x 103cfu of Mtb dispersed in 2 to 20 mL of water, urine, blood, or sputum. For the 20 mL dispersion, the separation was carried out by adding more MNP -Phage with a total mass of 300 pg (indicated by a star *). (FIG. 25C) Different numbers of Mtb mixed with l*106cfu of interfering bacteria (C. diphtherias, E. coli, S. pneumoniae, or S. aureus). A blank sample was prepared by adding 10 pL of ATP reagent in 100 pL PBS, whose bioluminescent signal was arbitrarily set as 1 to normalize the test samples. The solid line and the grey bar represent the signal of the blank sample plus three times the standard deviation. Unless marked with a star *, the mass of MNP -Phage added for BCG separation was 100 pg in each test.

[0041] FIGs. 26A-D show according to an exemplary embodiment of the invention a device and method of using the device for collection and processing of sputum samples using magnetic phages, which has five parts as shown in FIG. 26A as well as a Valve and Magnet. Part l is a lid, Part 2 is top container, Part 3 is a column, Part 4 is a container, and Part 5 is a container. The assembled device is shown in FIG. 26B, where the valve is closed, and both the sputum and magnetic phages are mixed in the top container part 2. Next, after the opening of the valve, magnetic T / A-phage complexes will be immobilized in column part 3 by the magnets (indicated as black lines), and the rest of the sputum solution will flow down to container part 4 (FIG. 26C). The sputum waste container part 4 will be replaced by a clean, smaller container 5 (FIG. 26D). After the removal of the magnets on column part 3, Mtb- a Q complexes will be collected into container part 5 for analysis.

[0042] DETAILED DESCRIPTION

[0043] Bacteriophages are bacterial viruses commonly present in the environment and can infect the target bacteria with high specificity. Biosensors constructed with bacteriophages enable the detection of many bacteria strains. For example, mycobacterium virus D29 is notable for its ability to infect viable Mtb cells to detect tuberculosis by plaque counting assays. Although the sensitivity is high, the entire assay must be performed at biosafety level 3 (BSL-3) laboratories, and the long turnaround time (~30 h) is unsatisfying. Combining the recognition capability of bacteriophages with magnetic nanotechnologies to achieve the enrichment and sensitive detection of Mtb is an alternative approach.

[0044] Magnetic nanoparticles (MNP) have been studied as efficient separation agents for biomedical applications. Smaller nanoparticles with diameters less than 30 nm have excellent colloidal stability and can remain homogenous suspension in complicated biological medium; however, these nanoparticles require a strong field provided by an electromagnet (2 Tesla) to perform desirable magnetic separation. On the contrary, micron or 2 submicron-sized magnetic beads that respond quickly to the permanent magnet (0.5 T) need complicated and heavy surface coatings to maintain uniform distributions in high salt conditions. The lengthy preparation process and high cost make them less competitive in large- scale productions.

[0045] Magnetic clusters are a novel magnetic nanomaterial formed by clustering nanocrystals (4~10 nm) into controlled aggregates (20-200 nm). They exhibit superior magnetic properties, such as large magnetizations and acute responsiveness to the external field. The primary nanocrystals are partially merged during synthesis, and the shared interfaces facilitate the alignment of the magnetic dipoles within the cluster in which a stronger magnetic moment is realized. Magnetic clusters can achieve rapid and efficient magnetic separation within several tens of minutes using a cheap, handheld permanent magnet.

[0046] Despite their strong magnetic moment, the clusters can maintain homogenous suspension in complicated media, making them suitable for applications in various environments. In this invention, conjugation of magnetic clusters with mycobacteriophage (MNP -Phage) was achieved to separate target cells with high specificity and efficiency. Using the luciferase reaction to generate strong bioluminescent signals, the magnetically enriched bacteria were quantified based on the adenosine triphosphate (ATP) content. Finally, rapid, point-of-care detection of tuberculosis in clinically relevant matrices was demonstrated, including sputum, urine, and blood, with a detection limit of 500 cfu / mL.

[0047] Phage functionalization and MNP conjugation

[0048] In one exemplary embodiment, the bacteriophage was propagated with its host bacteria, M. smegmatis (Msmeg). Its long-tail morphology was characterized using transmission electron microscopy (TEM) and is shown in FIG. 1. To achieve conjugation of bacteriophage with magnetic nanoparticles, the bacteriophage is firstly functionalized with dopamine, a linker molecule that can strongly bind with the iron oxide nanoparticle. The schematics are shown in FIG. 2. The catechol head groups of dopamine can form strong bidentate bonds with iron. Including an electron-withdrawing nitro functionality in this structure further strengthens the iron-catechol bond. The amine group of dopamine is used to form an amide bond with the carboxylic acid groups of the phage’s protein shell, thus covalently anchoring dopamine to bacteriophage (Dopa-Phage).

[0049] A series of Dopa-Phage samples were prepared by varying the amount of dopamine, as characterized by UV-Vis absorbance and plaque assay (FIGs. 3A- B). In PBS buffer, the bacteriophage has a single strong absorption at 270 nm. In the plaque assay, the bacteriophage induced the lysis of the host bacteria and formed uniform plaques on the agar plate. The phage concentration in the stock solution was calculated to be 1.5*10nplaque-forming units per milliliter (pfu / mL). Meanwhile, nitro-dopamine (10 pg / mL) has a unique absorption peak at 425 nm. After dopamine conjugation, these Dopa-Phage samples all demonstrated absorption around 425 nm. The dopa-to-phage ratio was measured around several tens to a hundred molecules per bacteriophage, with a maximum conjugation capacity of 150. The multiple grafting sites on the phage can ensure the MNP -Phage bond remains nearly irreversible. It was noticed that the dopamine-functionalized phage samples could not effectively induce the lysis of bacteria, indicating that the chemical alteration on the bacteriophage has impeded their life cycles and would be helpful for target pathogen enrichment due to their minimum disruption of the bacteria.

[0050] Following hydrothermal synthesis, the size-controlled magnetic clusters were prepared. Their morphologies are examined by TEM, as shown in FIG. 4. In contrast to conventional single-core nanoparticles, each cluster has hundreds of primary nanocrystals (~4 nm), and the cluster diameter is narrowly distributed with an average of 65 nm. The as-synthesized MNP is coated with polyacrylic acid, with negatively charged carboxylic acid groups providing excellent colloidal stability. Dynamic light scattering (DLS) characterization shows that the average hydrodynamic size of the MNP is 93 nm, as shown in FIG. 5. The difference between the hydrodynamic and physical sizes examined by TEM is due to the polyacrylate coatings extending to the solution from the MNP core. To anchor Dopa-Phage onto the MNP, 1011pfu Dopa-Phage (60: 1) was mixed with 2 mg of the as-synthesized MNP (65 nm clusters) in 10 mL water for two hours. In DLS, MNP -Phage is about 30 nm larger than the as-synthesized MNP. Furthermore, the zeta-potential of the MNP-4Phage (-33 mV) is almost comparable to as-synthesized MNP (-38 mV), indicating that the Dopa-Phage only partially replaced the original polyacrylate coatings of the MNP. As a result, the MNP -Phage has good colloidal stability and magnetic separation efficiency. With no external magnetic field, the MNP-Phage solution was stable for over three days, and the hydrodynamic size remained unchanged. The MNP- Phage can be efficiently separated using the magnet, and a complete magnetic separation only takes 20 minutes, as shown in FIG. 6. The magnetically collected MNP-Phage can be fully redispersed in water by simply shaking the solution. More importantly, MNP-Phage can be lyophilized for extended storage time. The dried powder can be homogeneously redispersed in water using a bath sonicator. At room temperature, the dried powers were stored on the shelf for three months, and no apparent decline in their performances was observed after being redispersed in water.

[0051] On the contrary, conjugating dopamine first with MNP can result in the crosslinking between the MNPs in the following EDC coupling reaction, as the MNP-dopamine contains both carboxylic acid groups from polyacrylate and amine groups from dopamine. Although directly coupling the carboxylic acid groups on the MNPs to the amine groups on phages can also form MNP-Phage conjugation, this method neutralizes the charges of polyacrylate. In both cases, the resultant MNP-Phage samples have poor colloidal stability.

[0052] Magnetic Separation of BCG

[0053] The as-prepared MNP-Phage was first examined to see its capability in targeting the host bacteria, Msmeg. An aliquot of a fresh culture containing 106cfu of Msmeg was dispersed in 10 mL water, followed by adding 100 pg MNP-Phage into the solution. The Msmeg and MNP-Phage mixture was incubated for one hour. The mixture was then magnetically washed three times to remove the nonmagnetic components and examined by TEM. As shown in FIG. 7A, the dark dots with an average diameter of 65 nm are the MNPs, and the pale rod-shaped objects about 2 to 5 pm in length and 0.4 to 0.6 pm in width correspond to the Msmeg. Auramine O was used to stain the magnetically collected content to confirm the presence of mycobacteria. Under the fluorescent microscope, a strong fluorescence was observed, as shown in FIG. 8. As Auramine O is positively charged, it induced the aggregation of MNPs, which along with the bacteria, formed larger rod shapes during magnetic separation.

[0054] As BCG and Mtb share the same peculiar cell wall structure of Msmeg, it is reasonable to suppose a similar affinity of the MNP-Phage to these mycobacteria. FIGs 7B-C show the TEM images of the MNP -Phage bound to BCG and Mtb. A comparable result was found that the BCG and Mtb cells were also surrounded by the MNPs, indicating that phages derived from Msmeg would be useful for tuberculosis detection. Additional TEM images (FIG. 9) were taken where fewer bacteria (105and 103cfu) were used. As there were relatively more particles than bacteria, the bacteria were heavily covered by multiple layers of MNP in these cases. The inventors estimated that each bacteria cell was bound with a few hundred particles. Moreover, similar phenomena were also observed for other non-tuberculosis mycobacteria (NTM), including M. avium, M. abscessus, and M. scrofulaceum, as shown in FIG. 10. The above results indicate the MNP -Phage’s high affinity to the mycobacterium genus.

[0055] In a control experiment, the as-synthesized MNP was also directly mixed in these bacteria dispersion, and no bacteria were captured and observed under the electron microscope. Since the bacteria cell wall and the as-synthesized MNP are both negatively charged, the two components are repulsive to each other. The adsorption of MNP to the surface of bacteria is unlikely to occur.

[0056] The workflow for the magnetic separation and enrichment of target bacteria was established. As illustrated in FIG. 11, in a small Eppendorf tube, a fresh culture of BCG (104cfu) and 100 pg MNP -Phage were dispersed in 1 mL PBS buffer. After incubating at room temperature with a rotation speed of 180 rpm for 20 min, the vial was placed in a magnet rack. The vertical arrangement of the magnets can prevent the unbound bacteria from falling into the magnetic separated content, which was collected on the side. While the MNP -Phage needed 20 min for a complete separation, those bound with bacteria took less: within 3 min, a complete magnetic separation was achieved with the colorless supernatant solution. This is due to the magnetic interactions among the particles bound to the bacteria cell, making them more magnetic responsive. The supernatant solution was collected while the magnetic content was redispersed in 1 mL PBS. Both solutions were then plated on the 7H10 agar plates. After five days of incubation at 37 °C, the magnetic content formed uniform spots on the agar plates, indicating that BCG remained viable after magnetic separation. On the contrary, no colonies were observed after plating the supernatant solution, indicating a nearly 100% magnetic separation of the target bacteria from the solution. The inventors attributed this excellent separation performance to the fact that the MNP -Phage can homogenously disperse in the solution, where they have much higher chances of contacting the bacteria and achieving successful binding.

[0057] Quantified Detection of BCG

[0058] An ATP reagent, CellTiter-Glo, is employed to quantify the numbers of captured bacteria with the MNP -Phage and examine the detection limit. The assay relies on cellular ATP to transform luciferin to oxyluciferin, releasing strong bioluminescent signals. However, due to the high concentrations of mycolic acid in the waxy lipid-rich outer layer of the mycobacteria cell walls, the ATP release is slow in aqueous solution. In 100 pL PBS solution with no lysis buffer, 104cfu of magnetically separated Msmeg, BCG, and Mtb were dispersed, followed by the addition of 10 pL of the ATP reagent. The result is shown in FIGs. 12A-B. The bioluminescent signal for Msmeg and BCG peaked around 10 min and was about ten times higher than the blank sample; the signal for Mtb reached a maximum after 20 min and was merely two times higher than the background. The slow release of the bacteria ATP led to an unsatisfied detection limit. Although integrating the signal over time would increase the sensitivity, this method would become more time-consuming and require an advanced luminometer for the programmed integration.

[0059] To facilitate the rapid release of bacteria ATP, the inventors relied on sonic energy to mechanically break up the cellular structures of these bacteria. In another set of experiments, the magnetically collected bacteria were first put in a bath sonicator for 15 seconds before adding the ATP reagent. The bioluminescent signals were immediately measured, as shown in FIGs. 12A-B, 13A-C: a five-to-six-fold increase in the signal intensity was found for Msmeg and BCG, and a ten-fold increase for Mtb. By breaking up the cells, the bioluminescent detection of bacteria was shortened to 2 min, with a much higher signal intensity achieved.

[0060] Other structural characterizations, including the MNP size and Dopa-to-Phage ratio, were also made to improve the detection sensitivity further. Larger magnetic clusters (120 nm) and smaller clusters (28 nm), as well as conventional single-core nanoparticles (12.5 nm), were prepared, as shown in FIG. 14. Using the handheld magnet, the time needed for a complete magnetic separation (>95%) of the 120 nm, 65 nm, and 28 nm clusters (100 pg / mL) are 1 min, 20 min, and 12 hours, respectively. In contrast, the 12.5 nm nanoparticles could not be magnetically separated under this condition, in which an electromagnet is needed. After conjugating these MNPs with Dopa-Phage, their hydrodynamic sizes were increased by 15 to 30 nm (FIG. 15). The bacteria- capturing performances of MNP -Phage samples were then compared using different-sized clusters or nanoparticles. After incubating 0 to 5*103cfu of Msmeg with 100 pg MNP -Phage, the vial was placed in the magnet rack for 20 min to collect the magnetic separation content, whose bioluminescent signals were then recorded, as shown in FIG. 16A. The low signal intensities for the 28 nm clusters and the 12.5 nm nanoparticles were due to their slow magnetic separation rate, in which a large portion of the bacteria had yet to be collected. Despite their fast separation, the 120 nm clusters have poor colloidal stability, limiting their accessible surface area for bacteria binding. Thus, the signal intensity was also not satisfying. Therefore, the 65 nm clusters were selected as the magnetic separation candidate.

[0061] The performances of the MNP -Phage samples prepared using the 65 nm clusters and Dopa-Phage with different dopa content were also compared, as shown in FIG. 16B. An intermediate Dopa-to-Phage ratio of 60: 1 was found to have the maximum signal intensities consistently. When there are too few dopamine molecules on the bacteriophage, the bounding between the MNP and phage is not strong enough. When there are too many, these dopamine molecules could impede the bacteria recognition abilities of the tail fiber proteins on the phage. Also, the excessive anchoring points to nanoparticles could induce the crosslinking of MNPs and phage, leading to undesired colloidal stability. An intermediate Dopa-Phage ratio ensures the optimal binding between the MNP and the phage, while the bacteria recognition function is minimally compromised after chemical modification.

[0062] After optimizing the MNP -Phage and detection method, a calibration curve for BCG detection was generated by recording the bioluminescent signal versus the total number of BCG used in magnetic separation. 102to 106cfu of BCG and 100 pg of MNP-Phage were first mixed in 1 mL PBS buffer. After 20 min incubation, BCG were magnetically separated, with the MNP -BCG pellet redispersed in 100 pL PBS. 10 pL of the ATP assay reagent was added after the sufficient sonication on the bacteria, and the bioluminescent signal was measured immediately. As shown in FIG. 13A, the curve has a great linear range between 103to 105cfu of BCG, with an acute detection limit of 350 bacilli in the initial solution. The calibration curves were also established for Msmeg and Mtb, as shown in FIGs. 17A-B, where a similar detection limit and linear range were found. The highly sensitive detection and good linearity make the MNP-Phage an excellent candidate for tuberculosis screening.

[0063] It is noticed that the curve growth rate slows down when the number of BCG exceeds 106cfu. To examine the bacteria enrichment capacity of the magnetic separation, 105and 104cfu of BCG were mixed with 10 to 200 pg of MNP- Phage. After magnetic separation, the bioluminescent signals on both the magnetically separated content and the supernatant solution were recorded and shown in FIG. 13B. With more MNP-Phage used, the bioluminescent signal of the magnetically separated content increased until the curve became saturated. Meanwhile, the signal on the supernatant decreased until it was below the detection limit. This phenomenon suggests that with enough MNP used for magnetic separation, BCG left in the supernatant solution was negligible, consistent with the results on the agar plates in FIG. 11. The enrichment capacity of MNP-Phage to BCG was estimated to be 1.5* 107cfu per milligram MNP, indicating an excellent recovery capacity using the phage-enabled magnetic separation of BCG. Magnetic separation and ATP assay detection was further performed on other bacterium strains, including M. avium, M. abscessus, M. scrofulaceum, C. diphtheriae, E. coli, K. pneumoniae, S. pneumoniae, and S. aureus. The result is shown in FIG. 13C. The strong bioluminescent signals from the mycobacterium strains suggest that MNP -Phage can also be employed to detect other diseases associated with NTMs. It was noted that the signal intensities were close to each other with the same numbers of bacteria, indicating a similar intracellular ATP concentration level in different bacteria strains. On the contrary, for strains other than mycobacterium, significant signals were not detected among them, suggesting the high specificity of the magnetic targeting, which can largely avoid false positive results in clinical tests.

[0064] The use of bacteriophage for magnetic separation of target bacteria can also be extended to other strains of bacteria. In another set of experiments with a different pair of bacterium and bacteriophage, E. coli and E. coli phage were cultured. The same conjugation procedure was applied to the E coli phage, and the MNP -Ephage was prepared. The result is shown in FIGs. 18A-C, where a detection limit of 2* 102cfu E. coli was found. Detection of Deactivated Bacteria

[0065] In clinical samples, the pathogenic bacteria might not be 100% viable. To gain insight into the affinity of the MNP -Phage to the nonviable bacteria, deactivated BCG were prepared by treating the actively growing BCG with methanol for 12 h. Deactivation was confirmed by the ATP assay, where no significant bioluminescent signal was detected. The deactivated BCG and MNP-Phage mixture was incubated and magnetically washed, and the bounding was examined by TEM. As shown in FIG. 19, despite the apparent distortions found in their cellular structures, the deactivated BCG were covered by the MNPs, suggesting the MNP-Phage can also separate the target bacteria regardless of their viability as long as relatively intact cellular structures were maintained.

[0066] To quantify the recovery efficiency of the deactivated BCG, a fluorescent probe was synthesized using FITC and 3 -aminophenyl boronic acid (FITC-APB). The staining of the fluorophore was established based on the strong interactions between the boric acid groups and glycolipids in bacteria cell membranes. Magnetically separated BCG using MNP-Phage was incubated with 10 pM FITC-APB for 30 min and then washed with PBS three times. These deactivated BCG under the fluorescent microscope (FIG. 20) exhibit strong fluorescence labeling. Further, the fluorescent signal intensities of different numbers of deactivated BCG that undergo magnetic separation and FITC-APB staining were measured, as shown in FIGs. 21A. The calibration curve indicates a moderate detection limit of 4*103cfu. It was also found that the fluorescent signals were comparable to the live BCG, suggesting the viability of the target bacteria has little effect on the detection sensitivity. Therefore, the fluorescent signal acquired in this detection assay could roughly correlate to the target bacteria's total numbers, whether live or dead. Finally, with a total of 104cfu, a series of bacteria samples with different viability were prepared by mixing the freshly prepared and deactivated BCG together and subsequently subject to bioluminescent and fluorescent detection after magnetic separation. As shown in FIG. 21B, the bioluminescent signals (left Y-axis) linearly increased as the viability increased from 0 to 1. In contrast, the fluorescent signals (right Y-axis) remained unchanged, corresponding to the constant total number of BCG. By performing both the ATP -based bioluminescent assay and the fluorescent detection assay and referring to the calibration curve, respectively, the viability of the target bacteria can be read out, which can provide useful clinical information for disease control and treatment.

[0067] Point-of-care Tuberculosis Detection in Clinical Conditions

[0068] To establish a point-of-care tuberculosis detection method and favor fast screening in resource-limited settings, we simplified the detection method using cost-effective and portable devices, as illustrated in FIG. 22A. These devices include a magnet rack for separation, a small ultrasonic cleaner for bacteria lysis, and a portable luminometer the size of a cellphone for signal readout. The ultrasonic cleaner and the luminometer are both battery-powered. MNP -Phage was first added in the test tubes with samples of bacteria suspension. After 20 min incubation at room temperature, these tubes were transferred to the magnet rack for magnetic separation and washed with PBS three times to remove the original medium and other impurities. The captured bacteria were then placed in a portable sonicator (10 W, 45 kHz) for 1 min. The elongated treatment time can compensate for the low power output and achieve a similar effect on bacteria lysis. Eventually, after adding the ATP reagent, the test tube was immediately transferred to the portable luminometer for signal readout. The signal readout of a blank sample, in which the ATP reagent was directly added to the PBS buffer, was set as 1.

[0069] The separation capabilities of the MNP -Phage in complicated medium and the potential in clinical applications was first examined; the inventors selected artificial urine, artificial sputum, and whole porcine blood to mimic the conditions in patient samples. Different numbers of BCG were dispersed in these solutions (1 mL) with the above detection flow performed to examine the detection sensitivity in clinical samples. To reduce the viscosity of the sputum and blood, the MNP-Phage was first dispersed in 1 mL PBS and then mixed with the sputum or blood sample. Diluting the sputum or blood with PBS can effectively facilitate the magnetic capture of the target bacteria. Eventually, the magnetically separated content was redispersed in 100 pL PBS buffer with 10 pL of the ATP assay reagent added after sonication. The result was compared with that in water, as shown in FIGs. 22A-E. The original dispersion medium had little effect on the signal readout. As low as 500 bacteria suspended in these media, the relative signal intensities were about 2.5-fold of the background, suggesting the excellent sensitivity of this detection method.

[0070] On the contrary, the bioluminescence was much lower without magnetic separation by adding the ATP reagent to these media (FIG. 23). Significant signals in blood and sputum were only found in much higher bacteria content (2* 103cfu), while in urine, there was no readout at all. As the salt concentration and the pH conditions do not favor the ATP-driven enzymatic reactions, removing the original medium and enriching the target bacteria in PBS contributes to the excellent sensitivity of this detection method. The bacteria capture and detection performance in other non-neutral pH solutions were also tested, and the result is shown in FIG. 24. MNP-Phage worked in both acidic (pH = 5) and basic (pH = 11) buffers, with similar signal intensities found in PBS. At more acidic conditions (pH = 3), the MNP-Phage aggregated, leading to the unsatisfied signal readout. However, this can be avoided by diluting the sample with PBS to adjust the pH value of the solution.

[0071] In clinical samples, the target bacteria concentrations can be extremely low. To further test the sensitivity of this detection method, l*103cfu of BCG were dispersed into larger volumes (2 to 20 mL) of water, urine, blood, and sputum to reduce the initial BCG concentration. Again, MNP -Phage was first dissolved in an equal volume of PBS to ensure sufficient magnetic separation before mixing with sputum or blood. The result (FIG. 22C) shows that magnetic separation can efficiently detect BCG with a concentration as low as 100 cfu / mL, provided with enough sample volumes. This indicates a great potential for patients with urinal tract infection (UTI) or blood infection at an early stage, where larger sample volumes can be acquired.

[0072] Different bacteria strains could be present in the same sample for patients with multiple pathogen infections. To see whether other non-mycobacteria could compromise the detection sensitivity, BCG were mixed with 1000-fold more interfering bacteria, including C. diphtheriae, E. coli, S. pneumoniae, and S. aureus (FIGs. 22D-E). In one experiment, 106cfu of interfering bacteria were mixed with different numbers of BCG, from 0 to 2* 103cfu. Magnetic separation of BCG was performed on these samples using MNP -Phage. When no BCG were added, the signal intensities were equivalent to the background, suggesting MNP-Phage could not separate the interfering bacteria. A similar detection sensitivity of 500 cfu of BCG was found to generate significant signals, and as the number of BCG increased, the signal intensities increased accordingly. In another experiment, l*103cfu BCG were mixed hundred- to thousand-fold interfering bacteria. The viability of the magnetically collected content remained unchanged regardless of the presence of these bacteria. Both experiments suggest that the interferences from other bacteria to the magnetic separation of BCG were minimal, and the viability of BCGcould be selectively read out in a bacteria mixture sample.

[0073] To further demonstrate the efficacy of the assay for detection of Mtb in clinics, we employed the attenuated H37Rv laboratory strain of Mycobacterium tuberculosis. The inventors tested MNP -Phage in artificial urine, sputum, and whole porcine blood samples, dispersing different amounts of attenuated Mtb H37Rv in each medium to assess detection sensitivity. As shown in FIG. 25A, as few as 1000 cfu of attenuated Mtb H37Rv was detectable (about a threefold increase over the background). Further tests in 2-20 mL sample volumes detected as low as 50 cfu / mL by increasing the mass of MNP -Phage (FIG. 25B) To assess the specificity of Mtb capturing, mixing Mtb with high concentrations (l*106cfu) of C. diphtherias. E. coli. S. pneumoniae, or S. aureus showed minimal interference (FIG. 25C).

[0074] Experimental Section

[0075] Reagents and chemicals

[0076] 3,4-dihydroxyphenethylamine hydrochloride (dopamine hydrochloride), hydrogen peroxide solution (H2O2, ACS grade, 30%), trisodium citrate dihydrate (Na3Cit2H2O, 99%), sodium hydroxide solution (NaOH, 1 M), hydrochloric acid (HC1, ACS grade, 37%), dichloromethane (DCM, ACS grade, 99.5%), and N, N-dimethylformamide (DMF, anhydrous, 99.8%) were purchased from Fisher Scientific. Ethylene glycol (anhydrous, 99.8%), iron(III) chloride hexahydrate (FeC13 -6H2O, ACS reagent, 97%), urea (ACS reagent, 99.0%), Poly(acrylic acid sodium salt) (average Mw -5,100), Iron(III) acetyl acetonate (97%), diethylene glycol (99%), 2-(N-

[0077] Morpholino)ethanesulfonic acid (MES, 99%), N-Ethyl-N'-(3- dimethylaminopropyl)carbodiimide hydrochloride (EDC, 99%), sodium borohydride (NaBH4, 99%), sodium nitrite (NaNO2, anhydrous, 97%), magnesium sulfate (MgSO4, anhydrous, 99.5%), sodium bicarbonate (NaHCO3, 99.7%), ethyl acetate (EtOAc, ACS grade, 99%), fluorescein 5(6)- isothiocyanate (FITC, 90%), 3-aminophenyl boronic acid (98%), N,N- diisopropylethylamine (DIPEA, 99%), and dimethyl sulfoxide (DMSO, 99.9%) were purchased from Sigma-Aldrich. Ultrapure water was obtained from the Millipore Milli-Q lab water system.

[0078] Bacteria and phage culturing

[0079] Mycobacterium smegmatis phage (ATCC 11759-B1), M. smegmatis (Trevisan) Lehmann and Neumann (ATCC 11759), M. tuberculosis variant bovis BCG (ATCC 35734), 5. aureus (ATCC 25923), and C. diphtheriae (ATCC 13812) were purchased from ATCC and used as is. Attenuated Mycobacterium tuberculosis strain H37Rv was kindly provided by Niaz Banaei’s lab (Stanford University). Mycobacterium avium, Mycobacterium abscessus, and Mycobacterium scrofulaceum was kindly provided by Matthew Bogyo’s lab.

[0080] Msmeg and phage culturing

[0081] Msmeg and Msmeg phage were cultured in liquid 7H9 / ADC medium (4.7 g / L 7H9 powder, 0.2% glycerol, and 10% ADC supplement). 200 pL of previously cultured broth or 2 pL frozen Msmeg was added in 2 mL Middlebrook 7H9 broth. The culturing tube was then placed in an incubator at 37 °C with a rotation speed of 180 rpm. The optical density of the culturing broth at 600 nm (OD600) was monitored on the UV-Vis spectrometer every 3 to 4 hours. The culture was stopped when OD600 reached around 0.5, and the culture broth was centrifuged at 10,000 rpm for 1 min to get rid of the broth solution. The bacteria pellet was redispersed in PBS buffer. The colony forming units per milliliter (cfu / mL) is estimated by the OD600 with a conversion of OD600 of 1 equal to 5*108cfu / mL.

[0082] An actively growing broth culture of Msmeg was first prepared to propagate phage. The OD600 was first adjusted to around 0.2, in which Msmeg were in the early log phase. Approximately 10 pg of the freeze-dried phage was dissolved in 100 pL 7H9 and then added to 2 mL of the Msmeg culture. The phage induced Msmeg was cultured at 37 °C with a rotation speed of 180 rpm for 2 days. The culturing solution was then centrifuged at 4,000 rpm for 10 min, and the supernatant was passed through a 0.22 pm syringe filter and washed with PBS buffer using a 100 kDa centrifuge filter unit.

[0083] Phage plaque assay

[0084] The plaque-forming units per milliliter (pfu / mL) of the collected phage was measured on a standard double-layer agar plate using the plaque assay protocols. The bottom hard agar (19.5 g / L 7H10 powder, 0.5% glycerol, and 10% OADC supplement) was first poured into the plate and solidified at 37 °C. The top soft agar (4.7 g / L 7H9 powder, 0.2% glycerol, 0.7% agar, 3*108cfu of early log phase Msmeg, and serial diluted phage solution) were then poured onto the hard agar layer. The plate was inverted and incubated at 37 °C for 3 days to count the number of plaques in each plate. The concentration of phage in the stock solution was around 1.5* 1011pfu / mL as determined by the plaque assay.

[0085] Other bacteria culturing

[0086] BCG were cultured in liquid 7H9 / ADC medium (4.7 g / L 7H9 powder, 0.1% Tween-80, 40mM sodium pyruvate, and 10% ADC supplement). Attenuated Mtb H37Rv was cultured in liquid 7H9 / ADC medium (4.7 g / L 7H9 powder, 0.2% glycerol, 2 g / L casamino acid, 24 mg / L pantothenate, 80 mg / L lysine, and 10% ADC supplement). Three non-tuberculosis mycobacteria (NTM), including M. avium, M. abscessus, M. scrofiilaceiim, were cultured in 7H9 / OADC medium (0.2% glycerol, 0.05% Tween-80, and 10% OADC supplement). E. coli was grown in LB medium. S. pneumoniae was grown in tryptic soy broth. S. aureus and C. diphtheriae were grown on tryptic soy agar plates supplemented with defibrinated sheep blood. K. pneumoniae was grown nutrient agar plate.

[0087] MNP synthesis and functionalization

[0088] MNP synthesis Magnetic clusters: In a typical synthesis, 540 mg FeC13 -6H2O, 250 mg polyacrylic acid (Mw~5,000), and 1200 mg urea were dispersed in 20 mL ethylene glycol, followed by 0 mg, 500 mg, and 1 g water for the 120 nm, 65 nm, 28 nm clusters, respectively. The mixture was then transferred to a Teflon-lined hydrothermal reactor and heated at 180 °C for 8 hours. After cooling down, the as-synthesized clusters were magnetically washed three times with water. As a comparison, the single-core magnetic nanoparticles were also prepared using thermal decomposition. S412.5 nm nanoparticles: 176 mg Iron(III) acetylacetonate [Fe(acac)3] was first dissolved in 5 mL diethylene glycol and heated to 230 °C. After 2 hours, 2.5 mL diethylene glycol containing 88 mg Fe(acac)3 was injected into the solution and heated at 230 °C for another 2 hours. After the heating was turned off, 2.5 mL di ethylene glycol containing 100 mg polyacrylic acid (Mw~5,000) was added dropwise. The reaction mixture was then dispersed in 20 mL water and washed using centrifugal filtration (30 kDa) three times. Nitro-dopamine synthesis

[0089] Dissolve 100 mg dopamine hydrochloride in 10 mL water and stir vigorously in an ice bath, followed by the addition of 130 mg sodium nitrite. Slowly add 1 mL 20% sulfuric acid to the mixture and vent the nitrogen dioxide gas from the reaction. Remove the ice bath and leave the mixture stirring at room temperature overnight. The reaction product, nitrodopamine hydrogensulfate, was purified by filtering the mixture and washing the residue with ice water three times. The residue was then freeze dried into a yellow powder. The reaction yield was nearly 100% based on the mass of the dried nitro-dopamine.

[0090] Dopa-Phage conjugation

[0091] In a typical conjugation reaction, 1 mg N-Ethyl-N'-(3- dimethylaminopropyljcarbodiimide hydrochloride (EDC) and 1011 pfu of previously collected phage was added in 1 mL MES buffer (100 mM) and was mixed for 5 min at room temperature to activate the carboxylic acid groups on phage. The mixture was then transferred in 20 mL 0.1 M carbonate-bicarbonate buffer (pH = 9.2), followed by adding 0.05, 0.1, 0.25, 0.5, 1 mg nitro-dopamine, respectively. The reaction mixture was allowed to undergo at room temperature for 2 h. The buffer solution is removed using a 100 kDa centrifuge filter unit. The concentrated content was then transferred to a dialysis membrane tubing (3 kDa) and dialyzed against water for 1 d to remove the excessive EDC and dopamine. The as-prepared dopa-phage conjugate was characterized by UV-Vis absorbance at 400 nm to determine the dopamine concentration.

[0092] Coating MNP with Dopa-Phage

[0093] To anchor Dopa-Phage onto the MNP, 1011pfu Dopa-Phage was mixed with 1~2 mg of the as-synthesized MNP (65 nm clusters) in 10 mL water for two hours at room temperature. The MNP -Phage was then magnetically washed three times to remove unbounded Dopa-Phage. The as-prepared MNP -Phage was passed through a 0.22 pm syringe filter to remove crosslinked particles.

[0094] Zeta potential and hydrodynamic size measurement

[0095] The zeta potential and hydrodynamic size of the nanoclusters with different coatings are measured on a Zetasizer Nano ZS (Malvern Instruments Ltd, Malvern, UK) at room temperature. All the colloidal solutions have a pH of 7. All samples are measured in triplicate. S5FITC-APB synthesis In 1 mL DMSO, 10 mg FITC (25.7 mM), 3.5 mg 3-aminophenyl boronic acid (25.7 mM), and 3.3 mg N,N-diisopropylethylamine was added. The reaction mixture was stirred at room temperature for 2 hours to allow the isothiocyanate group to react with the amine group. The product was purified by UPLC. Conclusion

[0096] In summary, rapid and cost-effective detection of tuberculosis through the conjugation of magnetic clusters with mycobacteriophage (MNP -Phage) to separate target cells was achieved. The bacteriophage propagated in Msmeg was functionalized with dopamine, which caused it to lose the ability to complete a lytic cycle. Electron microscopy images showed the strong binding of the mycobacteria with the nanoparticles coated with Dopa-Phage. The magnetically enriched bacteria maintained viable cellular structures and were disrupted by ultrasound to facilitate the rapid release of cellular ATP. The number of bacteria was quantified using the ATP assay to generate strong bioluminescent signals.

[0097] A high specificity toward mycobacteria species and nearly 100% separation efficiency were examined. Finally, portable and inexpensive devices were used, including a magnet rack, a small ultrasonic cleaner, and a luminometer, to demonstrate rapid, point-of-care tuberculosis detection in clinically relevant matrices, including sputum, urine, and blood, and with other interfering bacteria. A detection limit of 500 cfu / mL for BCG was achieved with a detection time of 35 min.

Claims

CLAIMSWhat is claimed is:

1. A bacteriophage functionalized with magnetic nanocrystal clusters, comprising:(a) a bacteriophage;(b) nitro-dopamine molecules conjugated to the bacteriophage; and(c) magnetic nanocrystal clusters anchored to hydroxyl groups of the nitro-dopamine molecules.

2. The bacteriophage as set forth in claim 1, wherein the bacteriophage is Mycobacterium smegmatis phage (ATCC 11759-B1) or E. coli phage.

3. The bacteriophage as set forth in claim 1, wherein the magnetic nanocrystal clusters range in size from 20 nm to 200 nm.

4. The bacteriophage as set forth in claim 1, wherein each of the magnetic nano-crystals is about 65 nm in size.

5. The bacteriophage as set forth in claim 1, wherein a ratio between the nitro-dopamine molecules to the bacteriophage is from 90: 1 to 20: 1.

6. The bacteriophage as set forth in claim 1, wherein a ratio between the nitro-dopamine molecules to the bacteriophage is about 60: 1.

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