Patent Application
20220011304
The present disclosure relates to methods and devices for detection of analytes. The methods and devices disclosed herein may be used for detection of whole-cell bacteria.
Incorporated by reference in its entirety herein is a computer-readable nucleotide/amino acid sequence listing submitted concurrently herewith and identified as follows: One 871 bytes ASCII (Text) file named “18-050-092012-9080-W001-SEQ-LIST-12-20-18.txt,” created on Dec. 20, 2018.
The worldwide development of multidrug resistance in microbes not only threatens public health globally, but also carries a hefty economic burden through increasing health care costs and loss of productivity. At least three different classes of antibiotics, including carbapenems, last resort antibiotics, have failed to treat multidrug resistant bacterial infections. In 2017, the World Health Organization listed carbapenem-resistant strains of Acinetobacter baumannii and Pseudomonas aeruginosa as top priority pathogens. Gram-negative bacterial infections are particularly challenging to treat due to: (a) a thick, altered cell wall structure which significantly reduces the permeability of drugs and diminishes drug binding efficiency, (b) production of enzymes to destroy antibiotics, and (c) evolved efflux mechanisms to out-pump antibiotics. To combat the burgeoning global crisis of multidrug resistant microbes, two parallel efforts are urgently needed: new antibiotics which circumvent the evolutionary potential of microbes to evade destruction, and advanced diagnostic tools which can specifically and sensitively detect the presence of pathogenic strains of microbes, thereby avoiding the over-prescription of wide-spectrum antibiotics.
Current gold standard microbiology methods for bacterial detection require culturing bacteria in a strictly controlled laboratory environment and often take days for an accurate reading. Immunoassays, an alternative strategy, are rapid but suffer from relatively low sensitivity with a typical limit of detection (LOD) of 103-106 cfu mL−1 (cfu=colony-forming unit). Other approaches like polymerase chain reaction (PCR) and its derivatives afford fast and sensitive detection but require extensive sample preparation to extract DNA from bacteria as well as expensive reagents and instrumentation. As such, methods for bacterial detection which optimize detection speed, sensitivity, selectivity, and cost for bacterial diagnostics are needed.
In one aspect, disclosed herein are biosensors for detection of analytes. The biosensors may be used for whole-cell bacterial detection. The biosensor may comprise an array of gold nanoparticles; biotinylated polyethylene glycol thiol in direct contact with one or more of the gold nanoparticles; polyethylene glycol thiol in direct contact with one or more of the gold nanoparticles; at least one neutravidin molecule immobilized on a surface of the biotinylated polyethylene glycol thiol; and at least one affinity reagent immobilized on a surface of the at least one neutravidin molecule. The affinity reagent may be a biotinylated aptamer. The affinity reagent may be a biotinylated siderophore. The disclosed biosensors may be used in a method of detecting bacterial infection in a subject. For example, the disclosed biosensors may be used in a method of detecting Pseudomonas aeruginosa infection or Acinetobacter baumannii infection in a subject.
Other aspects of the disclosure will become apparent by consideration of the detailed description and accompanying drawings.
Disclosed herein are biosensors and methods for detection of analytes in a sample. For example, the biosensors and methods may be used for whole-cell bacterial detection. The biosensors disclosed herein are characterized by rapid detection, extraordinary sensitivity, clinically relevant dynamic range, selectivity over detection of other bacteria, and/or excellent shelf life when stored in ambient conditions.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In case of conflict, the present document, including definitions, will control. Preferred methods and materials are described below, although methods and materials similar or equivalent to those described herein can be used in practice or testing of the present invention. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. The materials, methods, and examples disclosed herein are illustrative only and not intended to be limiting.
The terms “comprise(s),” “include(s),” “having,” “has,” “can,” “contain(s),” and variants thereof, as used herein, are intended to be open-ended transitional phrases, terms, or words that do not preclude the possibility of additional acts or structures. The singular forms “a,” “an” and “the” include plural references unless the context clearly dictates otherwise. The present disclosure also contemplates other embodiments “comprising,” “consisting of” and “consisting essentially of,” the embodiments or elements presented herein, whether explicitly set forth or not.
The modifier “about” used in connection with a quantity is inclusive of the stated value and has the meaning dictated by the context (for example, it includes at least the degree of error associated with the measurement of the particular quantity). The modifier “about” should also be considered as disclosing the range defined by the absolute values of the two endpoints. For example, the expression “from about 2 to about 4” also discloses the range “from 2 to 4.” The term “about” may refer to plus or minus 10% of the indicated number. For example, “about 10%” may indicate a range of 9% to 11%, and “about 1” may mean from 0.9-1.1. Other meanings of “about” may be apparent from the context, such as rounding off, so, for example “about 1” may also mean from 0.5 to 1.4.
For the recitation of numeric ranges herein, each intervening number there between with the same degree of precision is explicitly contemplated. For example, for the range of 6-9, the numbers 7 and 8 are contemplated in addition to 6 and 9, and for the range 6.0-7.0, the number 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are explicitly contemplated.
Acinetobacter baumannii and A. baumannii are used interchangeably herein. A. baumannii is a typically short, almost round, rod-shaped Gram-negative bacterium. It can be an opportunistic pathogen in humans, affecting people with compromised immune systems. Some possible types of A. baumannii infections include Pneumonia, bloodstream infections, meningitis, wound and surgical site infections, and urinary tract infections. Symptoms of A. baumannii infections include fevers and chills, rash, confusion and/or altered mental states, pain or burning sensations when urinating, strong urge to urinate frequently, sensitivity to bright light, nausea (with or without vomiting), muscle and chest pains, breathing problems, and cough (with or without yellow, green, or bloody mucus). In some cases, A. baumannii may present no infection or symptoms, as with colonizing an open wound or tracheostomy site.
“Affinity” and “binding affinity” as used interchangeably herein refer to the tendency or strength of binding between molecules. For example, affinity may refer to the tendency of strength of binding of avidin, streptavidin, or neutravidin to biotin. As another example, affinity may refer to the tendency of binding of an aptamer to its target bacterium. The binding affinity may be represented by the equilibrium dissociation constant (KD), the dissociation rate (kd), or the association rate (ka).
“Aptamer” as used herein refers to an oligonucleotide or peptide molecule that can bind to pre-selected targets with high affinity and specificity. The pre-selected target may be any desired bacteria, including Pseudomonas aeruginosa. Aptamers may assume a variety of shapes due to their propensity to form helices and single-stranded loops. An oligonucleotide or nucleic acid aptamer can be a single-stranded DNA or RNA (ssDNA or ssRNA) molecule. A peptide aptamer can include a short variable peptide domain, attached at both ends to a protein scaffold.
“Gram positive” is a taxonomic feature referring to bacteria which take up standard crystal violet primary Gram-staining dye. Gram staining is a process that differentiates bacteria by the chemical and physical properties of their cell walls by detecting peptidoglycan, which is present in the cell walls of Gram-positive bacteria. Gram-positive bacteria may have more extensive peptidoglycan crosslinking in their cell walls than Gram-negative bacteria.
“Gram-negative” is a taxonomic feature referring to bacteria which do not take up standard crystal violet primary Gram-staining dye. Peptidoglycan is typically present in a small layer in the cell walls of Gram-negative bacteria. This small layer is easily dissolved with certain organic solvents such as, for example, ethanol or acetone, thus preventing the cell from retaining the violet primary stain. Gram-negative cells may be characterized by their cell envelopes, which are composed of a thin peptidoglycan cell wall sandwiched between an inner cytoplasmic cell membrane and a bacterial outer membrane. The ability of bacteria to retain or resist staining generally reflects the structure of the cell wall, and it has been suggested that Gram-positive bacteria have more extensive peptidoglycan crosslinking and than their Gram-negative counterparts.
Pseudomonas aeruginosa and P. aeruginosa are used interchangeably herein. Pseudomonas aeruginosa is a common Gram-negative, rod-shaped bacterium that can cause disease in plants and animals. P. aeruginosa typically infects the airway, urinary tract, burns, and wounds, and also causes other blood infections. P. aeruginosa has many strains, including strain PAO1, PA7, UCBPP-PA14 (PA14), and Pseudomonas aeruginosa strain 2192. Some strains of P. aeruginosa have developed multi-drug resistance.
“Sample”, “biological sample”, or “test sample” as used interchangeably herein can mean any sample in which the presence and/or level of a target bacteria is to be detected. Samples may include a biological sample. Samples may include any biological fluid or tissue, such as blood, whole blood, fractions of blood such as plasma and serum, muscle, interstitial fluid, sweat, saliva, urine, tears, synovial fluid, bone marrow, cerebrospinal fluid, nasal secretions, sputum, amniotic fluid, bronchoalveolar lavage fluid, gastric lavage, emesis, fecal matter, lung tissue, peripheral blood mononuclear cells, total white blood cells, lymph node cells, spleen cells, tonsil cells, cancer cells, tumor cells, bile, digestive fluid, skin, or combinations thereof. In some embodiments, the sample comprises an aliquot. In other embodiments, the sample comprises a biological fluid. Samples can be obtained by any means known in the art. The sample can be used directly as obtained from a patient or can be pre-treated, such as by filtration, distillation, extraction, concentration, centrifugation, inactivation of interfering components, addition of reagents, and the like, to modify the character of the sample in some manner as discussed herein or otherwise as is known in the art.
“Siderophore” as used herein refers to a molecule that binds and transports iron. A siderophore may be a natural siderophore secreted by microorgansims such as bacteria or fungi. A siderophore may be a synthetic siderophore.
“Subject” and “patient” are used interchangeably herein. The subject may be a human or a non-human animal. The subject may be a vertebrate. The subject may be a mammal. The mammal may be a primate or a non-primate. The mammal can be a primate such as a human; a non-primate such as, for example, dog, cat, horse, cow, pig, mouse, rat, camel, llama, goat, rabbit, sheep, hamster, and guinea pig; or non-human primate such as, for example, monkey, chimpanzee, gorilla, orangutan, and gibbon. The subject may be male. The subject may be female. In some embodiments, the subject is human. The subject or patient may be undergoing other forms of treatment.
Disclosed herein are biosensors for detection of analytes. The biosensors disclosed herein may be used for whole-cell bacterial detection.
The biosensor comprises an array of gold nanoparticles. The array of gold nanoparticles may be immobilized on the biosensor surface. The gold nanoparticles may be immobilized on the biosensor surface by any suitable technique known in the art, for example by nanosphere lithography (NSL). The array of gold nanoparticles may comprise any suitable shape of nanoparticle, including but not limited to triangles, spheres, rods, stars, cubes, octahedrons, plates, and prisms. For example, the array of gold nanoparticles may comprise gold nanotriangles.
The gold nanoparticles may be of any suitable size useful for analyte detection. For example, the gold nanoparticles may have an in-plane width of about 10 nm to about 500 nm. The gold nanoparticles may have an in-plane width of about 10 nm to about 500 nm, about 25 nm to about 450 nm, about 50 nm to about 400 nm, about 75 nm to about 350 nm, about 100 nm to about 300 nm, about 125 nm to about 250 nm, or about 150 nm to about 200 nm. For example, the gold nanoparticles may have an in-plane width of about 180 nm, about 190 nm, about 200 nm, about 210 nm, about 220 nm, or about 230 nm. The gold nanoparticles may have an out-of-plane height of about 20 nm to about 60 nm. For example, the gold nanoparticles may have an out-of-plane height of about 20 nm, about 30 nm, about 40 nm, about 40 nm, or about 60 nm.
The biosensor may comprise gold nanoparticles aligned in any suitable array for analyte detection. For example, the biosensor may comprise a hexagonal array of gold nanoparticles. The gold nanoparticles in the array may be spaced at any suitable interparticle distance to useful for whole-cell bacterial detection. For example, the gold nanoparticles may be spaced at an interparticle distance of about 50 nm to about 300 nm. For example, the gold nanoparticles may be spaced at an interparticle distance of about 50 nm to about 300 nm, about 75 nm to about 250 nm, about 100 nm to about 200 nm, or about 125 nm to about 150 nm. For example, the gold nanoparticles may be spaced at an interparticle distance of about 100 nm, about 110 nm, about 120 nm, about 130 nm, or about 140 nm.
The biosensor further comprises biotinylated polyethylene glycol thiol in direct contact with one or more of the gold nanoparticles. The biosensor further comprises polyethylene glycol thiol in direct contact with one or more of the gold nanoparticles. The biosensor may comprise a ratio of biotinylated polyethylene glycol thiol to polyethylene glycol thiol of about 1:10 to about 1:1 by volume. For example, the biosensor may comprise a ratio of biotinylated polyethylene glycol thiol to polyethylene glycol thiol of about 1:3 by volume.
The biosensor may further comprise a molecule with high affinity for biotin immobilized on a surface of the biotinylated polyethylene glycol thiol. For example, the biosensor may comprise at least one neutravidin molecule immobilized on a surface of the biotinylated polyethylene glycol thiol. As another example, the biosensor may comprise at least one avidin molecule immobilized on a surface of the biotinylated polyethylene glycol thiol. In some embodiments, the biosensor may comprise at least one streptavidin molecule immobilized on a surface of the biotinylated polyethylene glycol thiol.
The biosensor may further comprise at least one affinity reagent immobilized on a surface of the at least one molecule with high affinity for biotin. The affinity reagent may be any suitable affinity reagent that selectively binds to the desired analyte in a sample. The analyte may be a bacterium, fungus, virus, and the like. For example, the affinity reagent may be any suitable affinity reagent that selectively binds to a bacterial cell in a sample. The bacterial cell may be a whole bacterial cell.
In some embodiments, the affinity reagent is an aptamer. The aptamer may be biotinylated. For example, the biosensor may further comprise a biotinylated aptamer immobilized on a surface of the at least one molecule with high affinity for biotin. For example, the biosensor may comprise at least one biotinylated aptamer immobilized on a surface of the neutravidin molecule. The biotinylated aptamer may be designed or selected to bind to any desired target analyte. For example, the biotinylated aptamer may be designed or selected to bind to any desired target bacteria. In some embodiments, the biotinylated aptamer may be designed or selected to bind to whole cell bacteria. For example, the biotinylated aptamer may be designed or selected to bind to Gram-positive bacteria. As another example, the biotinylated aptamer may be designed or selected to bind to Gram-negative bacteria. In some embodiments, the biotinylated aptamer may be selected to bind to Pseudomonas aeruginosa. The biotinylated aptamer may selectively bind Pseudomonas aeruginosa. For example, the biotinylated aptamer may bind Pseudomonas aeruginosa without appreciable binding to other bacterial strains. The biotinylated aptamer may bind Pseudomonas aeruginosa without appreciable binding to other Gram-negative bacteria. For example, the biotinylated aptamer may bind Pseudomonas aeruginosa without appreciable binding to E. coli. The biotinylated aptamer may bind Pseudomonas aeruginosa without appreciable binding to Gram-positive bacteria. For example, the biotinylated aptamer may bind Pseudomonas aeruginosa without appreciable binding to S. aureus. The biotinylated aptamer may selectively bind Pseudomonas aeruginosa strain PAO1. For example, the biotinylated aptamer may selectively bind Pseudomonas aeruginosa strain PAO1 without appreciable binding to other Pseudomonas aeruginosa strains, such as PA14. In some embodiments, the biotinylated aptamer may selectively bind Pseudomonas aeruginosa strain PAO1 without appreciable binding to Pseudomonas aeruginosa strain PA14, E. coli, or S. aureus.
In some embodiments, the biotinylated aptamer is encoded by a nucleotide sequence with at least 80% sequence identity with SEQ ID NO: 1. For example, the biotinylated aptamer may be encoded by a nucleotide sequence with at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity with SEQ ID NO: 1. In some embodiments, the biotinylated aptamer is encoded by the nucleotide sequence of SEQ ID NO: 1.
In other embodiments, the affinity reagent may be a siderophore. The siderophore may be a natural siderophore or an artificial siderophore. In some embodiments, the siderophore is biotinylated. For example, the biosensor may comprise at least one biotinylated siderophore immobilized on a surface of the neutravidin molecule. The siderophore may be a hydroxamate siderophore. The siderophore may be a catecholate siderophore. The siderophore may be a biscatecholate-monohydroxamate mixed ligand siderophore. The biscatecholate-monohydroxamate mixed ligand siderophore may comprise three repeating polyethylene glycol units linked to a biotin molecule.
The siderophore may selectively bind any desired target analyte. For example, the siderophore may selectively bind any desired target bacteria. In some embodiments, the siderophore may selectively bind to whole cell bacteria. The siderophore may selectively bind Acinetobacter baumannii. For example, the biotinylated siderophore may bind Acinetobacter baumannii without appreciable binding to other bacterial strains. The biotinylated siderophore may bind Acinetobacter baumannii without appreciable binding to other Gram-negative bacteria. For example, the biotinylated siderophore may bind Acinetobacter baumannii without appreciable binding to E. coli or Pseudomonas aeruginosa. The biotinylated siderophore may bind Acinetobacter baumannii without appreciable binding to Gram-positive bacteria. For example, the biotinylated siderophore may bind Acinetobacter baumannii without appreciable binding to B. cereus. In some embodiments, the biotinylated aptamer may selectively bind Acinetobacter baumannii without appreciable binding to Pseudomonas aeruginosa, E. coli, and B. cereus.
The disclosed biosensors demonstrate surprisingly high stability. The disclosed biosensors may be stable in ambient conditions for at least 2 weeks. For example, the disclosed biosensors may be stable in ambient conditions for at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, at least 7 weeks, at least 8 weeks, at least 9 weeks, at least 10 weeks, at least 12 weeks, at least 4 months, at least 5 months, or at least 6 months. In some embodiments, the disclosed biosensors are stable in ambient conditions for at least 2 months. The disclosed biosensors also demonstrate high stability when stored in N2 environment. When stored in N2 environment, the disclosed biosensors may be stable for at least at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, at least 7 weeks, at least 8 weeks, at least 9 weeks, at least 10 weeks, at least 12 weeks, at least 4 months, at least 5 months, or at least 6 months. In some embodiments, the disclosed biosensors are stable in N2 environment for at least 2 months.
The disclosed biosensors demonstrate surprisingly high sensitivity. The disclosed biosensors may be used to detect whole-cell bacteria wherein the concentration of bacteria in a sample is less than or equal to about 4×106 cfu/mL. For example, the disclosed biosensors may be used to detect whole-cell bacteria when the concentration of bacteria in a sample is about 4×106 cfu/mL, less than about 4×106 cfu/mL, less than about 4×105 cfu/mL, less than about 4×104 cfu/mL, less than about 4×103 cfu/mL, less than about 1000 cfu/mL, less than about 100 cfu/mL, or less than about 10 cfu/mL. In some embodiments, the disclosed biosensors may be used to detect a single bacterium in a sample, indicating extraordinary sensitivity for whole-cell bacterial detection.
Further disclosed herein are methods for detecting bacterial infection in a subject. The method comprises obtaining a sample from the subject and contacting the sample with a biosensor disclosed herein.
The sample may be contacted with the disclosed biosensor under any suitable conditions for any suitable duration of time to achieve the desired binding of whole cell bacterial cells to the biosensor. In some embodiments, the sample may be contacted with the disclosed biosensor at about 37° C. for about 1 hour. The sample may be contacted with the biosensor under controlled humidity conditions.
After being contacted with the sample for the desired duration of time, the biosensor may be washed in any suitable buffer to remove bacterial cells that did not bind to the biosensor. When dried, salt crystals on sensor surfaces, can contribute to an apparent LSPR wavelength shift, thus confounding the bacterial-derived signal. In addition, lysis of bacteria could give rise to LSPR signals not correlated with the number of bacteria present in the sample. Thus, it is preferable to wash the biosensor under conditions sufficiently stringent to minimize salt crystal formation, yet gentle enough to leave surface-captured bacteria intact. For example, the biosensor may be washed three times with undiluted PBS for 5 minutes each wash. After washing in PBS, the biosensor may be immersed in water. For example, the biosensor may be washed three times for 5 minutes each with undiluted PBS, followed by water immersion for 30 seconds. The biosensor may be dried prior to characterization of LSPR spectra. For example, the biosensor may be dried with N2 gas for 5 minutes prior to characterization of LSPR spectra.
The method further comprises detecting localized surface plasmon resonance (LSPR) of the sample and a control sample. The method further comprises comparing the wavelength shift of the maximum extinction of the LSPR of the sample to a LSPR wavelength shift in a control sample. Detecting the wavelength of the maximum extinction of the LSPR of the sample and/or that of the control sample may be performed using any suitable method under any suitable conditions known in the art. In some embodiments, UV-visible extinction spectroscopy may be used to detect the wavelength of the maximum extinction of the LSPR of the sample and/or that of the control sample. In some embodiments, UV-visible extinction spectrum measurements may be performed under controlled humidity conditions as the disclosed biosensor chips may be sensitive to humidity and temperature. In some embodiments, UV-visible extinction spectrum measurements may be performed in air. In other embodiments, UV-visible extinction spectrum measurements may be performed in suitable buffer, such as PBS.
Detecting bacterial infection using the disclosed biosensors involves measuring the wavelength shift (Δλ) before and after capture of the analyte, such as a whole bacterial cell, which subsequently causes a change in the dielectric environment, as reflected in the refractive index (n), surrounding the nanoparticles. This relationship can be expressed phenomenologically as Δλ=mΔn [1−exp (−2d/ld)] where m is the refractive index sensitivity, Δn is the change in refractive index induced by an adsorbate, d is the effective thickness of the adsorbate layer, and ld is the characteristic decay length for the evanescent electromagnetic field.
In accordance with the disclosed methods of detecting bacterial infection in a subject, a shift in the wavelength of maximum extinction of the localized surface plasmon resonance (LSPR) of the sample relative to the shift in the wavelength of maximum extinction of the LSPR of the control sample indicates a bacterial infection in the subject. In some embodiments, a concentration of bacteria of less than or equal to about 4×106 cfu/mL produces a positive shift in the wavelength of maximum extinction of the LSPR of the sample, indicating bacterial infection in the subject. For example, a concentration of bacteria of about 4×106 cfu/mL, less than about 4×106 cfu/mL, less than about 4×105 cfu/mL, less than about 4×104 cfu/mL, less than about 4×103 cfu/mL, less than about 1000 cfu/mL, less than about 100 cfu/mL, or less than about 10 cfu/mL may produce a positive shift in the wavelength of maximum extinction of the LSPR of the sample, indicating bacterial infection in the subject. In some embodiments, the disclosed biosensors may be used to detect bacterial infection in a subject when less than 100 bacterial cells are present in the sample. For example, the disclosed biosensors may be used to detect bacterial infection in a subject when less than 100, less than about 90, less than about 80, less than about 70, less than about 60, less than about 50, less than about 40, less than about 30, less than about 20, less than about 10, or less than about 5 bacterial cells are present in the sample. In some embodiments, the disclosed biosensors may be used to detect bacterial infection in a subject when a single bacterium is present in the sample, demonstrating the extraordinary sensitivity of the disclosed biosensors.
The disclosed method may detect bacterial infection in the subject in less than 24 hours. For example, the disclosed method may detect bacterial infection in the subject in less than 24 hours, less than about 12 hours, less than about 6 hours, less than about 5 hours, less than about 4 hours, or less than about 3 hours. In some embodiments, the method may detect bacterial infection in the subject in approximately 3 hours.
The disclosed method may detect Gram-positive bacterial infection in the subject. Gram-positive bacteria include, for example, Actinomyces spp. (A. israelii); Aerococcus spp.; Bacillus spp. (B. anthracis); Bacterionema spp.; Bifidobacterium spp.; Clostridium spp. (C. botulinum, C. difficile, C. perfringens, C. tetani); Corynebacterium spp. (C. diphtherias); Corprococcus spp.; Deinobacter spp.; Deinococcus spp.; Enterococcus spp. (E. faecalis, E. faecium); Erysipelothrix spp.; Eubacterium spp.; Gemella spp.; Lactobacillus spp.; Lactococcus spp.; Leuconostoc spp.; Listeria spp. (L. monocytogenes); Marinococcus spp.; Melissococcus spp.; Methanobacterium spp.; Micrococcus spp.; Mycobacterium spp. (M. avium, M. leprae, M. lepromatosis, M. tuberculosis, M. ulcerans); Micropolyspora spp.; Nocardia spp. (N. asteroides); Pediococcus spp.; Peptococcus spp.; Peptostreptococcus spp.; Planococcus spp.; Propionibacterium spp.; Rothia spp.; Ruminococcus spp.; Saccharococcus spp.; Salinococcus spp.; Carcina spp.; Staphylococcus spp. (S. aureus, S. epidermidis, S. saprophyticus); Stomatococcus spp.; Streptococcus spp. (S. agalactiae, S. pneumoniae, S. pyogenes, S. viridans); Streptomyces spp.; Trichococcus spp.; and Vagococcus spp. The subject may be infected with or at risk of infection from any combination of Gram-positive bacteria.
The disclosed method may detect Gram-negative bacterial infection in the subject. Gram-negative bacteria include, for example, Acinetobacter spp. (A. baumannii); Bacterioides spp. (B. fragilis); Bordetella spp. (B. pertussis); Borrelia spp. (B. burgdorferi, B. garinii, B. afzelii); Brucella spp. (B. abortus, B. canis, B. melitensis, B. suis); Burkholderia spp. (B. mallei, B. pseudomallei); Calymmatobacterium spp.; Campylobacter spp. (C. fetus, C. jejuni); Chlamydia spp. (C. trachomatis, C. pneumoniae, C. psittaci); Chlamydophila spp. (C. pneumoniae); Citrobacter spp.; Coxiella spp. (C. burnetti); Edwardsiella spp.; Enterobacter spp.; Ehrlichia spp. (E. canis, E. chaffeensis); Escherichia spp. (E. coli); Francisella spp. (F. tularensis); Gardnerella spp.; Haemophilus spp. (H. influenzae); Helicobacter spp. (H. pylori); Klebsiella spp. (K. pneumoniae); Legionella spp. (L. pneumophila); Leptospira spp. (L. interrogans); Moraxella spp. (M. catarrhalis); Mycoplasma spp. (M. pneumoniae); Neisseria spp. (N. gonorrhoeae, N. meningitidis); Pasteurella spp.; Proteus spp.; Providencia app.; Pseudomonas spp. (P. aeruginosa, P. mallei); Rickettsia spp. (R. akari, R. prowazekii, R. rickettsia); Salmonella spp. (S. enterica, S. enterica enteritidis, S. enterica hadar, S. enterica Heidelberg, S. enterica infantis, S. enterica paratyphi, S. enterica typhi, S. enterica typhimurium); Serratia spp.; Shigella spp. (S. dysenteriae, S. sonnei); Spirillaceae spp. (S. minus); Streptobacillus spp. (S. moniliformis); Treponema spp. (T. pallidum); Vibrio spp. (V. cholerae); and Yersinia spp. (Y. enterocolitica, Y. pestis, Y. pseudotuberculosis). The subject may be infected with or at risk of infection from any combination of Gram-negative bacteria.
In some embodiments, the disclosed method may detect Pseudomonas aeruginosa infection in the subject. In some embodiments, the disclosed method may detect infection with Pseudomonas aeruginosa strain PAO1 in the subject. In other embodiments, the disclosed method may detect Acinetobacter baumannii infection in the subject.
The biosensors and methods of the invention will be better understood by reference to the following examples, which are intended as an illustration of and not a limitation upon the scope of the invention.
The following Examples are offered as illustrative as a partial scope and particular embodiments of the disclosure and are not meant to be limiting of the scope of the disclosure.
Materials: Biotin PEG thiol (MW 1000 Da) was purchased from Nanocs. PEG thiol (MW 550 Da) was purchased from Creative PEGWorks. Neutravidin (NA) and tris(2-carboxyethyl)phosphine hydrochloride (TCEP) were purchased from Thermo Fisher Scientific. Polystyrene beads (0.6 μm mean particle size), sulfuric acid (95.0-98.0%), hydrogen peroxide (30% w/w in H2O), and ethyl alcohol (200 proof) were purchased from Sigma-Aldrich. Paraformaldehyde (16% w/v aqueous solution, methanol free), Dulbecco's Phosphate Buffered Saline (1×, without calcium and magnesium), and micro cover glass (No. 1, 18 mm×18 mm) were purchased from VWR. Lysogeny broth and granulated agar were purchased from BD Difco. Biotinylated aptamer (Bt-aptamer, 5′ [Bt] ATACCAGCTTATTCAATTCCCCCGTTGCTTTCGCTTTTCCTTTCGCTTTTGTTCGTTTC GTCCCTGCTTCCTTTCTTGAGATAGTAAGTGCAATCT3′) (SEQ ID NO:1) and fluorescently labeled biotinylated aptamer (6FAM-aptamer-Bt, 5′ [6-carboxyfluorescein-ATACCAGCTTATTCAATTCCCCCGTTGCTTTCGCTTTTCCTTTCGCTTTTGTTCGTTTC GTCCCTGCTTCCTTTCTTGAGATAGTAAGTGCAATCT-[Bt]3′) (SEQ ID NO: 2) were synthesized by Sigma-Aldrich. Deionized (DI) water used in all experiments was prepared using a Milli-Q Gradient water purification system with a resistivity of 18.2 MΩ·cm at 25° C. (Millipore).
Bacterial Cell Culture, Fixation, and Counting. P. aeruginosa strains PAO1 and PA14, Escherichia coli DH5α, and Staphylococcus aureus RN4220 used in these experiments were obtained from J. Shrout's laboratory (University of Notre Dame). All bacteria were streaked from freezer stocks and grown on lysogeny broth (LB) agar plates in a 37° C. incubator for 20 h. Cell cultures were first grown in a glass tube containing 6 mL of LB media for 9 h in a 37° C. incubator with shaking set at 240 rpm. Nine-hour bacterial samples (30 μL) were removed and serially diluted three times. All four serial dilutions were incubated at a 37° C. incubator for 14 h with shaking set at 240 rpm. All bacteria were harvested in their exponential growth phase. The cell culture was then washed twice using phosphate buffered saline (PBS) solution, and its optical density at 600 nm (OD600) was adjusted to 0.80. A serial dilution was carried out to achieve various bacterial concentrations. To inactivate bacterial cells, 0.7 mL of bacterial solution was added into a microcentrifuge tube containing 0.7 mL of 6% paraformaldehyde solution, prepared by diluting 16% paraformaldehyde solution in PBS. As a control, 0.7 mL of PBS solution was added to a microcentrifuge tube containing 0.7 mL of 6% paraformaldehyde solution. Both tubes were heated at 62.5° C. water bath for 1 h to inactivate bacteria in solution. All samples were cooled to room temperature prior to use. All bacterial cell densities were confirmed using standard microbiology plating and counting methods. Specifically, the OD600 of bacterial solution was adjusted to 0.80 upon harvesting cell culture. Each bacterial solution was diluted 6 orders of magnitude in LB media, and 100 μL of diluted bacterial solution was added onto a LB agar plate. The bacterial solution was spread evenly on a LB agar plate using a metal spreader, which was dipped in ethanol and flamed before and after each use. The bacteria containing LB agar plates were then incubated overnight at 37° C., and the colonies were counted the next day. Bacterial cell density was obtained by averaging the counts from nine LB agar plates (three plates were prepared in one experiment, and the same experiment was repeated three times on different days).
Sensor Fabrication. LSPR sensor chips were fabricated using NSL. Briefly, glass slides were soaked in piranha solution (Caution: piranha, 3:1 sulfuric acid/hydrogen peroxide, is a strong oxidizer and should be used with extreme caution!) overnight and rinsed with water. Polystyrene (PS) beads were gently spread as a monolayer at an air-water interface. The PS bead monolayer was transferred by first immersing a clean glass slide through the monolayer at an oblique angle, then translating the slide laterally to transfer PS beads onto the glass surface. The PS bead coated glass slides were dried at 60° C. prior to electron beam evaporation (UNIVEX 450B, Oerlikon) of Cr (1 nm) and Au (50 nm). The slides were then sonicated in chloroform to remove the PS beads, rinsed with water, dried with N2 gas, and stored under N2 environment. Each Au nanotriangle patterned glass slide (76.2 mm×25.4 mm) was diced into six pieces (10.16 mm×15.24 mm) using a dicing saw (Disco DAD3240) equipped with a diamond blade (Thermocarbon).
Sensor Surface Modification. A 1:3 (v:v) mixture of 1 mM Bt-PEG thiol:1 mM PEG thiol was self-assembled onto the sensor surface with mild shaking for 16 h. Then, 0.2 mL of neutravidin solution (1 mg/mL) was added with shaking for 1 h. Bt-aptamer solution (0.2 mL per tube) was heated in a 95° C. water bath for 30 min, cooled to room temperature, and then incubated with the neutravidin-modified sensor chips for 1 h with mild shaking under controlled humidity conditions. Sensor chips were gently rinsed with water and dried with N2 gas after each step.
Bacterial Detection. In a typical experiment, one chip was exposed to 0.2 mL of control solution, and five chips were exposed to 0.2 mL of inactivated bacterial solution under controlled humidity conditions. Control and bacterial samples were incubated at 37° C. for 1 h. Each chip was then washed three times in PBS solution for 5 min per wash, followed by water wash for 30 s. All sensor chips were dried with N2 gas setup for 5 min prior to characterization.
Characterization. LSPR spectra were acquired on a UV-visible/NIR spectrometer (Jasco V-670) equipped with a 60 mm integrating sphere (Jasco ISN-723). Each spectrum was averaged over three spectral accumulations collected from 400-1400 at 0.5 nm interval, and all extinction spectra were normalized. The probe beam size was ca. 8 mm×9 mm. For LSPR measurements in liquid, ˜50 μL of PBS solution was added onto the sensor surface, and a coverslip was added to confine the liquid prior to measurement. Scanning electron microscope images of Au nanotriangle arrays with and without bacteria were collected using a field-emission scanning electron microscope (FEI Magellan 400) at 5.00 kV. Prior to imaging, a thin layer of iridium (2.5 nm) was sputtered on the substrate to avoid surface charging.
A schematic illustration of a Au nanotriangle array, its layer-by-layer surface construction, and the bacterial detection scheme are shown in
Au nanotriangle arrays were modified by self-assembly of (1:3) Bt-PEG thiol: PEG thiol directly onto the Au surface of the nanotriangles. Tetrameric neutravidin (NA) was then linked to the surface bound biotin through its strong noncovalent interaction (Ka˜1013 M−1). PEG thiol was specifically chosen to repel nonspecifically adsorbed molecules and bacteria. The molecular weight difference between Bt-PEG thiol (MW 1000 Da) and PEG thiol (MW 500 Da) results in different chain lengths, facilitating the longer chain Bt-PEG thiol in its function to capture NA. The 1:3 (v:v) ratio (Bt-PEG thiol:PEG thiol) was selected, after careful optimization experiments, shown in
UV-visible extinction spectroscopy has been used to characterize LSPR resonances of Au nanoparticles both in solution and on surfaces and has been used to study molecular interactions at surfaces based upon LSPR wavelength shifts upon binding. It is well established that the refractive index of the surrounding environment affects the wavelength of the LSPR. To ascertain the optimal conditions for the aptamer-based assays, the sensitives of LSPR measurements in liquid (buffer) and air were compared by measuring the LSPR wavelength shift (Δλ) at several points in the assembly and capture process in both PBS and air. Measurements after immobilization of Bt-PEG thiol/PEG thiol, neutravidin, and Bt-aptamer onto the Au nanotriangle array, shown in
av = 17 for 103 cfu mL−1; v = 18 for 107 cfu mL−1
Here, bacterial pull-down was monitored by careful measurements of the UV-visible extinction spectrum. Care was taken to perform the measurements under controlled humidity conditions because the LSPR sensor chips are potentially sensitive to humidity and temperature.
When exposing the sensor surface to bacteria, it is important to simultaneously minimize salt crystal formation and avoid lysis of surface bound bacteria. When dried, salt crystals on sensor surfaces,
The sensitivity of sensor chips was evaluated by exposing them to various concentrations of P. aeruginosa and monitoring the LSPR wavelength shift (Δλ). Before evaluating sensitivity, it is important to understand the various contributions to sample-to-sample variability, which were compared using interbatch and intrabatch variability. Each batch consisted of a microscope slide diced into six small pieces: one chip as a control and the other five chips for testing separate bacterial samples. Each bacterial concentration was studied with 9-14 replicates. The interbatch variation was tested using two (or three in the case of 10 cfu mL−1) microscope slides fabricated in different batches but exposed to the same bacterial concentration. The interbatch variation was analyzed using the difference of means, which ranged from 0.2-4.0 nm (
Single bacterium experiments performed at 10 cfu mL−1 gave rise to a wavelength shift of =8.9±3.9 nm, which is statistically different than the control and the 1 cfu mL−1 result at the 99.9% confidence level. This exquisite sensitivity may originate from: (i) the inherent sensitivity of the LSPR sensor responding to small refractive index changes near the Au nanotriangle array surface, combined with the relatively thin capture layer which brings bacteria closer to the LSPR sensing volume, or (ii) a single bacterial cell on surface could distort local electric field around Au nanotriangles and provided an extraordinarily large signal. The LSPR wavelength shift saturated at concentrations higher than 103 cfu mL−1, although as suggested by the inset
LSPR-based sensors are not inherently selective; their selectivity comes from the choice of affinity reagent, in the present case, an aptamer specially selected to recognize fixed P. aeruginosa cells. Very few Pseudomonas-specific aptamer sequences are available, and the aptamer used in this study was previously identified by selection against inactivated P. aeruginosa using whole-cell SELEX, although the molecular nature of the interaction between the aptamer and the P. aeruginosa cell envelope has not been delineated. The selectivity of the aptamer was tested with two different strains of P. aeruginosa (PAO1, PA14), E. coli DH5α, and S. aureus RN4220 using the LSPR-based sensing platform. All four bacteria were harvested in the exponential growth phase (
E. coli
S. aureus
at-value for S. aureus at 99% level, others 99.9%; v = 14 (control); 16 (PA14 and S. aureus); 15 (E. coli)
The stability of the sensor chip in ambient conditions is important to its potential use in the field and in point-of-care applications. An ideal sensor chip would be stable over a wide range of temperatures and humidities for a long period of time. In this study, the stability of unmodified, Bt-PEG thiol/PEG thiol (1:3), and Bt-PEG thiol/PEG thiol (1:3)/neutravidin modified sensor chips was monitored for 2 months in ambient conditions by following the shifts in the extinction maxima.
An LSPR sensing platform was developed for the detection of whole-cell microorganisms, using P. aeruginosa strain PAO1 as a model system. The sensor chip was designed to pull down whole-cell P. aeruginosa cells from solution via a surface confined aptamer. Capture of bacteria was monitored through the resulting red-shift in wavelength of the LSPR extinction maximum. This LSPR whole-cell microbial sensing platform is (a) rapid (˜3 h), (b) highly sensitive, down to the level of a single bacterium, (c) selective against other strains of Pseudomonas and E. coli, and (d) stable in ambient conditions for >2 months. The general whole-cell LSPR sensing scheme demonstrated here could be translated to the detection of other microorganisms, including other bacteria as well as viruses, by switching affinity agents. This robust LSPR sensing platform has the potential to be used in clinical diagnostics and therapeutics, including in-the-field detection and point-of-care applications.
Materials: PEG thiol (MW 550 Da) was purchased from Creative PEGWorks. Biotin PEG thiol (MW 1000 Da) was purchased from Nanocs. NeutrAvidin Protein and formaldehyde (16% w/v aqueous solution, methanol-free) were purchased from Thermo Fisher Scientific. HOOK™ Biotin-PEG3-Amine was purchased from GBiosciences. Iron (III) acetylacetonate (Fe(acac)3, 99.9% trace metals basis), 2,2′-bipyridyl, tris(2-carboxyethyl)phosphine hydrochloride powder, isobutyl chloroformate (ClCO2i-Bu), tetrahydrofuran (THF), polystyrene beads (0.6 μm mean particle size), N-methylmorpholine, Mueller Hinton Broth, sodium phosphate monobasic monohydrate, sodium phosphate dibasic, ammonium phosphate dibasic, sulfuric acid (95.0 to 98.0%), hydrogen peroxide (30% w/w in H2O), dimethyl sulfoxide (DMSO), and ethyl alcohol (200 proof) were all purchased from Sigma-Aldrich. Glass microscope slides (3×1″) were purchased from Ted Pella. Micro cover glass (No. 1, 18 mm×18 mm) was purchased from VWR. Lysogeny broth and granulated agar were purchased from BD Difco. Deionized (DI) water was generated using a Milli-Q Gradient water purification system (Millipore, 18.2 MΩ·cm at 25° C.). Sterile 10 mM sodium phosphate buffer (pH 7.5) was used in all experiments. Tetrahydrofuran (THF) was freshly distilled over sodium before use. All other chemicals were used as received.
Siderophore Synthesis. All reactions were conducted under an atmosphere of dry argon unless otherwise stated. The mixed-ligand siderophore 1 (Schematic 1) was prepared. Specifically, to a cooled (0° C., ice-salt bath) solution of the penta-acetylated siderophore 1 (73 mg, 0.078 mmol) in anhydrous THF (1.5 mL) was added N-methylmorpholine (10.30 μL, 0.094 mmol, 1.2 equiv.) followed by isobutyl chloroformate (12.2 μL, 0.094 mmol, 1.2 equiv.). A white precipitate formed gradually after addition of the chloroformate (N-methylmorpholine hydrochloride salt). The reaction mixture was stirred at 0° C. and the progress of the isobutyl chloroformate ester formation was monitored by thin-layer chromatography (TLC) analysis. After 1 hour, TLC showed complete consumption of starting siderophore 1. The reaction mixture was filtered through a plug of cotton and rinsed with anhydrous THF (0.5 mL) to obtain a clear solution of the mixed anhydride of 1.
To conjugate PEG and biotin to the siderophore 1, the following procedures were carried out. In a separate round-bottom flask, biotin-PEG-amine (25 mg, 0.060 mmol, 0.77 equiv.) was added into a cooled (0° C., ice-salt bath) solution of sodium bicarbonate (15.7 mg, 0.187 mmol, 2.4 equiv.) in distilled DI water (2 mL). The mixed anhydride 1 was added to the resulting solution in a dropwise fashion over a period of 5 min. The reaction mixture was then stirred for 3 h at 0° C. (ice-salt bath). The coupling reaction progress, as indicated by the consumption of the biotin-PEG-amine, was monitored by reverse phase TLC and LC-MS analysis. The organic layer was separated, washed with brine, and concentrated to obtain the desired PEG-biotinylated siderophore as a mixture of products. The mixture was then purified by reverse phase chromatography (C18) using a gradient of CH3CN/H2O (20% to 70%) to obtain the desired biotinylated siderophore 2 (Schematic 1) as the major component. The minor components resulted from the loss of one (M-OAc), two (M-2OAc), three (M-3OAc) or four (M-4OAc) acetyl groups, and were also characterized by LC-MS analysis.
Preparation of 0.01 mg/mL Biotinylated Siderophore Solution. Biotinylated siderophore (Bt-siderophore) freezer stock (1 mg/mL) was prepared by dissolving 1.4 mg of synthesized Bt-siderophore into a mixture of 5% DMSO and 95% phosphate buffer saline solution to reach a final volume of 1.4 mL. Small aliquots of Bt-siderophore solution (0.01 mg/mL) were prepared by adding a 15 μL of Bt-siderophore (1 mg/mL) into a 74 μL of DMSO and a 1411 μL of 10 mM sodium phosphate solution. The small aliquots were mixed thoroughly and stored at −80° C. Each aliquot of Bt-siderophore solution (0.01 mg/mL) was slowly thawed at room temperature prior to use.
Preparation of Mueller-Hinton (MH) Iron Deficient Media. Mueller-Hinton (MH) media was prepared by dissolving 10.5 g of MH powder in 0.5 L of DI water. The MH media was autoclaved and cooled to room temperature. 2,2′-bipyridyl solution (1 mg/mL, 1 mL) was added into a 40 mL of cooled MH media. The MH media containing 2,2′-bipyridyl (MH iron deficient media) was stored at room temperature for at least 2 weeks prior to use, allowing 2,2′-bipyridyl to completely chelate environmental Fe (III) in solution and resulting in an iron deficient media.
Bacterial Cell Culture and Counting. Acinetobacter baumannii ATCC 17961 was obtained from Professor M. Miller's laboratory (University of Notre Dame). Pseudomonas aeruginosa strain PAO1, Escherichia coli DH5α, Bacillus cereus, and Staphylococcus aureus RN4220 were obtained from Professor J. Shrout's laboratory (University of Notre Dame). All bacteria were streaked from −80° C. freezer stocks and grown on lysogeny broth (LB) agar plates in a 37° C. incubator for 17 h. Liquid cell cultures were grown in MH iron deficient media. Specifically, cell cultures were first grown in a glass tube containing 6 mL of MH iron deficient media for 9 h in a 37° C. incubator with shaking at 240 rpm. Nine-hour bacterial cultures (30 μL) were removed and added to glass tubes containing 6 mL of MH iron deficient media. Diluted bacterial samples were grown at 37° C. for 12 h with shaking at 240 rpm prior to harvest. Cell cultures were washed twice using 10 mM sodium phosphate solution by centrifuging at 1000×G for 5 min each, and finally resuspended in sodium phosphate solution. The optical density of the resuspended cell culture was measured at 600 nm (OD600) and adjusted to 0.80. Serial dilutions were then performed using 10 mM sodium phosphate buffer to achieve desired bacterial concentrations. Diluted bacterial samples were used immediately.
Standard microbiology plating and counting methods were used to correlate bacterial cell density (cfu mL−1) to optical density (OD600). Specifically, the optical density of all bacterial solution was adjusted to 0.80 after resuspending bacteria in a 10 mM sodium phosphate solution. A. baumannii, P. aeruginosa, and E. coli solutions were diluted 6 orders of magnitude in a 10 mM sodium phosphate solution, while B. cereus solution was diluted 5 orders of magnitude due to the difference in cell density according to our preliminary studies. Diluted bacterial solution (100 μL) was added onto an LB agar plate and spread using a metal spreader, which was sterilized by dipping in ethanol and flamed before and after each use. The bacterial containing LB agar plates were incubated at 37° C. overnight, and colonies were counted the next day. Final bacterial cell density was calculated by averaging the colony counts from 9 LB agar plates (3 plates were prepared in one experiment, and the same experiment was repeated 3 times on different days). At OD600 of 0.80, there are 0.4×109, 1.1×109, 0.5×109, and 0.1×109 cfu mL−1 of A. baumannii, P. aeruginosa, E. coli, and B. cereus, respectively.
LSPR Sensor Chip Fabrication. LSPR sensor chips were fabricated via NSL. Briefly, glass microscope slides were immersed in piranha solution (Cation: piranha, a strong oxidizer, contains sulfuric acid/hydrogen peroxide in a 3:1 v/v and should be handled with extreme caution!) overnight, rinsed, and stored in DI water. Polystyrene (PS) beads were first diluted in ethanol (1:2 v/v) and well mixed. A monolayer of diluted PS beads was spread at an air-water interface and transferred onto a clean microscope slide by emersion through the interface. PS bead-coated glass slides were dried at 60° C., and then coated with Cr (1 nm) and Au (50 nm) using an electron beam deposition (UNIVEX 450B, Oerlikon). Metal coated glass slides were sonicated in chloroform for 15 min to remove the PS beads, resulting in a hexagonal nanoscale array of Au trigonal prisms on glass, and then rinsed with DI water, dried with N2 gas, and stored under N2 environment. A dicing saw (Disco DAD3240) equipped with a diamond blade (Thermocarbon) was used to cut each Au-patterned glass slide (76.2 mm×25.4 mm) into six individual sensor chips (10.16 mm×15.24 mm). All sensor chips were subsequently sonicated in ethanol and DI water for 5 min each, rinsed with DI water, dried with N2 gas, and stored under N2 environment prior to use.
Sensor Surface Modification. Each sensor chip was modified with a mixture of Bt-PEG thiol (1 mM) and PEG thiol (1 mM) in a 1:3 v/v ratio for 16 h with mild shaking. A volume of 0.2 mL of NeutrAvidin (1 mg/mL), Bt-siderophore (0.01 mg/mL), and Fe(acac)3 (1 mM) were subsequently incubated on the sensor surface for 0.5 h in a humidity-controlled environment with mild shaking. Sensor chips were rinsed with DI water and dried with N2 gas after each surface modification step. Fe(acac)3 solution was freshly prepared in 1% of DMSO and 9% of 10 mM ammonium phosphate dibasic solution (pH 8) prior to each experiment.
Bacterial Binding and Fixation. Bacterial binding experiments were carried out by adding 0.2 mL of bacterial solution at the desired concentration onto the siderophore-Fe (III) modified sensor chips. The bacterial solution used for all experiments contained bacteria in 10 mM sodium phosphate buffer. Control chips were exposed to 0.2 mL of 10 mM sodium phosphate solution. These sensor chips were incubated in a 37° C. incubator for 1 h in a humidity controlled environment, allowing bacteria to recognize and bind to surface-confined siderophore-Fe (III) complexes. To remove unbound bacterial cells, the solution on the sensor chips was gently decanted, and the sensor chips were then washed three times in 10 mM sodium phosphate (5 min per wash). To inactivate surface bound bacterial cells, 200 μL of 6% formaldehyde solution was added to the bacteria-containing sensor chips and incubated at 37° C. for 1 h in a humidity controlled environment. After 1 h cell fixation, the solution on the sensor chips was gently decanted, and the sensor chips were then washed in three times 10 mM sodium phosphate solution (5 min per wash), followed by a DI water wash for 30 s. Sensor chips containing bound inactivated bacteria were gently dried with N2 gas for 5 min prior to characterization.
Characterization. A UV-visible-NIR spectrometer (Jasco V-670) with a 60 mm integrating sphere (Jasco ISN-723) was used to acquire LSPR spectra. Each spectrum was an average of three spectral accumulations from 600-1400 at 0.5 nm interval. All extinction spectra were normalized. The probe beam size was ca. 8 mm×9 mm. Scanning electron microscope images of a bare sensor chip (i.e., nanoscale Au trigonal prism arrays) and a modified sensor chip with captured A. baumannii cells were acquired using a field-emission scanning electron microscope (FEI Magellan 400) at 5.00 kV. To avoid surface charging, a thin layer of iridium (2.0 nm) was sputtered on the substrate prior to imaging.
Liquid chromatography-mass spectrometry (LC-MS) was used to characterize both the freshly synthesized biotinylated siderophore (Bt-siderophore) and the Bt-siderophore in a buffer solution. In general, a UPLC system, autosampler, and photodiode array detector (Dionex Ultimate 3000) were coupled to a quadrupole time-of-flight hybrid mass spectrometer (Bruker MicrOTOF-Q II). Analytes were separated using a Thermo Scientific Acclaim™ RSLC 120 C18 column (2.2 μm particle size, 120 Å pore size, 2.1 mm inner diameter, 100 mm length) with a mobile phase composed of either A=0.1% formic acid in water or B=0.1% formic acid in acetonitrile. The samples were eluted at 0.4 mL/min with a mobile phase gradient of (i) 90% A/10% B for 2 min, (ii) 0% A/100% B for 18 min, (iii) 90% A/10% B for 2 min. For the freshly synthesized Bt-siderophore, the eluted samples were ionized in positive ion mode using a Bruker electrospray ionization source (end plate offset voltage=−500 V, capillary voltage=2200 V, N2 as a nebulizer at 5 bar and a dry gas at 10.0 L/min flow rate at 220° C.). Mass spectra were acquired over a mass range of 50-3000 Da. For the Bt-siderophore in buffer, the eluted samples were ionized in positive ion mode using a Bruker electrospray ionization source (end plate offset voltage=−500 V, capillary voltage=2400 V, N2 as a nebulizer at 4 bar and a dry gas at 7.0 L/min flow rate at 180° C.). Mass spectra were acquired at 5000 scans/s over a mass range of 300-3000 Da. Hystar 3.2 software was used in both data analyses.
Thin layer chromatography of freshly synthesized biotinylated siderophore. Sorbent Technologies silica gel 60 (32-63 μm) was used for all silica gel column chromatography purifications. Reverse phase chromatographic purifications were performed on Teledyne Instruments' 30 gram RediSep Rf Gold® C18Aq reversed-phase columns (column volume: 26.4 mL, average particle size: 20-40 average pore size: 100 Å) at a flow rate of 35 mL/min. Thin layer chromatography (TLC) was performed with Al-backed Merck 60-F254 or Al-backed Merck RP-C18 F256 silica gel plates using a 254 nm lamp and aqueous FeCl3 for visualization.
The key molecular recognition motif, a biscatecholate-monohydroxamate mixed ligand siderophore with three polyethylene glycol (PEG) repeating units linked to a biotin (Bt) (Compound 2, Schematic 1) was synthesized as described above. Briefly, the penta acetyl-protected glutaryl siderophore 1 was converted to the corresponding chloroformate active ester by treatment with isobutyl chloroformate (ClCO2i-Bu) in the presence of N-methylmorpholine (NMM) in THF at 0° C. The chloroformate ester formation was considered complete when TLC analysis showed complete consumption of the starting siderophore 1. The PEG-Bt-siderophore, 2, was obtained by coupling the mixed anhydride to the biotin-PEG-amine in aqueous bicarbonate, and purified using reverse phase chromatography C18 columns with a gradient of CH3CN/H2O. LC-MS analysis of the freshly synthesized biotinylated siderophore showed a major component and four minor components. The minor components resulted from the loss of one (M-OAc), two (M-2OAc), three (M-3OAc), and four (M-4OAc) acetyl groups. Just prior to bacterial binding experiments, the biotinylated siderophore in buffer was also characterized using LC-MS.
The LC-MS analysis of the freshly synthesized biotinylated siderophore showed that there were a major component and four minor components in the synthesized biotinylated siderophore 2 (Schematic 1). The major component, [m+2H]2+ calculated for C63H91N9O21S, had a m/z value of 671.8110, which matched to its theoretical value (671.8098). The minor components resulted from the loss of one (M-OAc), two (M-2OAc), three (M-3OAc), and four (M-4OAc) acetyl groups. The experimental and theoretical m/z values of these minor components were: 650.8031 and 650.8045 for [m+2H]2+ calculated for C61H89N9O20S (M-OAc), 629.8050 and 629.7992 for [m+2H]2+ calculated for C59H87N9O19S (M-2OAc), 608.7950 and 608.7939 for [m+2H]2+ calculated for C57H85N9O18S (M-3OAc), as well as 587.7968 and 587.7887 for [m+2H]2+ calculated for C55H83N9O17S (M-4OAc). Additionally, LC-MS analysis of the biotinylated siderophore in buffer (
Sensor Chip Fabrication and Sensor Surface Modification. A schematic illustration of the siderophore-based whole-cell LSPR sensor chip is shown in
The sensor surface was first modified with a mixture of Bt-PEG thiol (1000 Da) and PEG thiol (550 Da) in a 1:3 v/v ratio via self-assembly. The PEG thiol, used to prevent nonspecific adsorption, has a shorter chain length than the Bt-PEG thiol used to anchor subsequent surface immobilized moieties. The 1:3 ratio was determined previously to produce maximum bacterial binding on the sensor surface. Tetrameric NeutrAvidin, with two biotin binding sites each on opposite sides of the protein, was subsequently immobilized onto the biotin modified sensor surface via the strong non-covalent biotin-NeutrAvidin linkage. Specifically, the affinity equilibrium constant, Ka, of NeutrAvidin for surface immobilized biotin is ˜5.5×1011M−1. NeutrAvidin, a deglycosylated avidin, was chosen over avidin and streptavidin due to its similar biotin-binding affinity, more neutral isoelectric point (pI 6.3), as well as its lower tendency for nonspecific binding.
The Bt-siderophore, 2, was subsequently immobilized onto NeutrAvidin modified sensor surface as an affinity reagent to pull-down A. baumannii. The demonstrated selectivity and potency recommend this siderophore as a promising affinity reagent. In order to be recognized and utilized by bacteria, a siderophore has to chelate Fe (III) to form a siderophore-Fe (III) complex presumably in a 1:1 ratio. Thus, Fe(acac)3 was subsequently introduced to the Bt-siderophore modified sensor surface as an additional iron source to saturate surface-bound siderophore. Bacteria-containing solutions were then exposed to the siderophore-Fe (III) modified sensor surface. A representative SEM image of surface captured A. baumannii cells is shown in
LSPR spectroscopy was used to characterize surface modifications and bacterial binding. Representative NIR extinction spectra of a bare sensor chip (curve 1), a modified sensor chip (curve 2), and a sensor chip with captured bacteria (curve 3) are shown in
Control Experiments. Various control experiments were carried out to investigate the role of both Bt-siderophore and externally added Fe (III) source in A. baumannii binding experiments.
The sensitivity of the siderophore biosensor was investigated as a function of A. baumannii concentration ranging from 4×102 to 4×106 cfu mL−1 in 10 mM sodium phosphate solution, with each bacterial concentration being tested using 10-17 individual sensor chips. The mean LSPR wavelength shift (Δλ) was measured to be 2.9±2.0 to 7.4±4.2 nm across this range, with the control sample showing −0.9±2.4 nm (
Selective detection of the target analyte is a crucial figure of merit for biosensors. The selectivity of the siderophore biosensor was assessed against both Gram-negative (Acinetobacter baumannii ATCC 1796, Pseudomonas aeruginosa strain PAO1, E. coli DH5α) and Gram-positive bacteria (Bacillus cereus). All four bacteria were grown in an iron deficient media, and bacterial concentrations were adjusted to ˜107 cfu mL−1 in 10 mM sodium phosphate solution. Control experiments were carried out using 10 mM sodium phosphate solution without bacteria. S. aureus, another Gram-positive bacterium, was tested but is not included in this study due to its inability to grow in the iron deficient media.
P. aeruginosa
E. coli
B. cereus
A. baumannii
a)
at-value for all bacteria at a 99.9% confidence level; n = 16 (A. baumannii); 10 (control); 12 (P. aeruginosa, E. coli, and B. cereus).
bControl sample presents the siderophore surface recognition agent, but has not been exposed to bacteria.
Stability. Biosensor stability over a wide range of conditions (e.g., temperature, humidity) is important for applications, especially in resource limited settings. To test the stability of our siderophore biosensor, the siderophore-Fe (III) modified sensor chips were stored in ambient conditions and their LSPR wavelength shifts were monitored every week for a month.
Versatility. The utility of the whole-cell LSPR sensing approach was extended in this study using the novel siderophore bacterial pull-down strategy. Results from these two systems suggest that the whole-cell LSPR sensing platform is versatile and could be extended to a wide range of super-molecular analytes (e.g., bacteria, viruses, fungi) by swapping target-specific affinity reagents. Thus, the whole-cell bacterial pull-down biosensor offers the opportunity to expand the diagnostic tool box for use against infectious agents.
It is understood that the foregoing detailed description and accompanying examples are merely illustrative and are not to be taken as limitations upon the scope of the invention, which is defined solely by the appended claims and their equivalents.
Various changes and modifications to the disclosed embodiments will be apparent to those skilled in the art. Such changes and modifications, including without limitation those relating to the chemical structures, substituents, derivatives, intermediates, syntheses, compositions, formulations, or methods of use of the invention, may be made without departing from the spirit and scope thereof.
For reasons of completeness, various aspects of the present disclosure are set out in the following numbered embodiments:
A biosensor for detection of analytes, the biosensor comprising:
The biosensor of embodiment 1, wherein the array of gold nanoparticles comprises gold nanotriangles.
The biosensor of embodiment 1 or 2, wherein the biosensor comprises a hexagonal array of gold nanoparticles.
The biosensor of any one of embodiments 1-3, wherein the biosensor comprises biotinylated polyethylene glycol thiol and polyethylene glycol thiol in a ratio of about 1:3 by volume.
The biosensor of any one of embodiments 1-4, wherein the affinity reagent is a biotinylated aptamer.
The biosensor of embodiment 5, wherein the biotinylated aptamer selectively binds Pseudomonas aeruginosa.
The biosensor of embodiment 6, wherein the biotinylated aptamer selectively binds Pseudomonas aeruginosa strain PAO1.
The biosensor of embodiment 7, wherein the biotinylated aptamer is encoded by the nucleotide sequence of SEQ ID NO: 1.
The biosensor of any one of embodiments 1-4, wherein the affinity reagent is a biotinylated siderophore.
The biosensor of embodiment 9, wherein the biotinylated siderophore is a biscatecholate-monohydroxamate mixed ligand siderophore.
The biosensor of embodiment 10, wherein the biscatecholate-monohydroxamate mixed ligand siderophore comprises three repeating polyethylene glycol units linked to a biotin molecule.
The biosensor of any one of embodiments 9-11, wherein the biotinylated siderophore selectively binds Acinetobacter baumannii.
A method of detecting one or more whole bacterial cells in a sample, the method comprising contacting the sample with the biosensor of any one of embodiments 1-12.
A method of detecting a bacterial infection in a subject, the method comprising:
The method of embodiment 14, wherein the array of gold nanoparticles comprises gold nanotriangles.
The method of embodiment 14 or 15, wherein the biosensor comprises a hexagonal array of gold nanoparticles.
The method of any one of embodiments 13-16, wherein the biosensor comprises biotinylated polyethylene glycol thiol and polyethylene glycol thiol in a ratio of about 1:3 by volume.
The method of any one of embodiments 13-17, wherein the affinity reagent is a biotinylated aptamer.
The method of embodiment 18, wherein the biotinylated aptamer selectively binds Pseudomonas aeruginosa.
The method of embodiment 19, wherein the biotinylated aptamer selectively binds Pseudomonas aeruginosa strain PAO1.
The method of embodiment 20, wherein the biotinylated aptamer is encoded by the nucleotide sequence of SEQ ID NO: 1.
The method of any one of embodiments 13-17, wherein the affinity reagent is a biotinylated siderophore.
The method of embodiment 22, wherein the biotinylated siderophore is a biscatecholate-monohydroxamate mixed ligand siderophore.
The method of embodiment 23, wherein the biscatecholate-monohydroxamate mixed ligand siderophore comprises three repeating polyethylene glycol units linked to a biotin molecule.
The method of any one of embodiments 22-24, wherein the biotinylated siderophore selectively binds Acinetobacter baumannii.
The method of any one of embodiments 13-25, wherein a concentration of bacteria of about 4×106 cfu/mL or less produces a positive shift in the wavelength of maximum extinction of the localized surface plasmon resonance of the sample, indicating bacterial infection in the subject.
The method of any one of embodiments 13-26, wherein the method detects a bacterial infection in the subject in approximately 3 hours.
The method of any one of embodiments 13-27, wherein the bacterial infection in the subject is a Pseudomonas aeruginosa infection or an Acinetobacter baumannii infection.
This application claims priority from U.S. Provisional Patent Application No. 62/774,542, filed on Dec. 3, 2018, the entire disclosure of which is herein incorporated by reference for all purposes.
This invention was made with government support under grant 1R01AI113219-01 funded by the National Institute of Health. The government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2018/066884 | 12/20/2018 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 62774542 | Dec 2018 | US |
