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WO2006104700A1 - Nanocapteurs de proteines magnetiques et leurs procedes d'utilisation - Google Patents

Nanocapteurs de proteines magnetiques et leurs procedes d'utilisation Download PDF

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WO2006104700A1
WO2006104700A1 PCT/US2006/009487 US2006009487W WO2006104700A1 WO 2006104700 A1 WO2006104700 A1 WO 2006104700A1 US 2006009487 W US2006009487 W US 2006009487W WO 2006104700 A1 WO2006104700 A1 WO 2006104700A1
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sensor
analyte
protein
tail
phage
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Edward H. Goldberg
Robert P. Guertin
Timothy P. Harrah
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Tufts University
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Tufts University
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G01N33/54326—Magnetic particles

Definitions

  • This application related to a nanosensor that incorporates a magnetic particle on a protein rod for the detection of analytes in a liquid sample.
  • biosensors capable of selectively detecting specific biomolecules in a sample population. Examples of such populations range from cells and model organisms for pharmaceutical and genome research to samples of the environment for pathogens and biological- warfare-agent detection. In the case of genome research, it is necessary to discover gene sequences that provide a blueprint of the cell or organism through systematic identification of known and predicted genes. It is also of great interest to use so-called genomic arrays for gene expression monitoring and for screening for sequence variants or mutations. For these applications, it would be desirable to survey greater than 1 Mbit of genomic information on a single chip that is only a few cm 2 in area. In contrast, biosensors used to screen pathogens may have relaxed requirements on the number of different biomolecules that must be sensed simultaneously, while placing greater emphasis, e.g., on detection time, minimum detection levels, field durability, overall system size, energy requirement and cost.
  • Bioreceptors that have been used as sensing elements include biomolecules, such as antibodies, enzymes, and nucleic acids.
  • biomolecules such as antibodies, enzymes, and nucleic acids.
  • the information collected by the sensing element regarding the receptor-target attachment must be converted into a signal that can be easily measured.
  • transduction mechanisms that can be exploited for converting this attachment information, including optical, electrochemical, magnetic, and mass sensitive measurements.
  • the choice of the particular bioreceptor/transducer combination will ultimately impact biosensor figures of merit, such as detection sensitivity, selectivity, repeatability, integrability, scalability, energy requirement and cost.
  • a bead-array-counter biosensor uses DNA- functionalilized magnetic nanoparticles as the target probe and complementary DNA-covered magnetoresistive materials as the receptor/transducer (Baselt, D.R. et al. Biosensors & Bioelectronics 1998, 13, 731-739).
  • target and receptor DNA hybridize, the magnetic particles bind to the sensing element and modify the local magnetic field. This change is measured by monitoring the electrical resistance of the element, where the resistance is proportional to the number of hybridized beads on the element.
  • the characteristic time scale of the Brownian relaxation can be determined directly by alternating current susceptibility measurements as a function of frequency, as the peak in the imaginary part of the alternating current susceptibility shifts to lower frequencies upon binding a target molecule to a magnetic nanoparticle; this frequency shift is consistent with an increase in the hydrodynamic radius corresponding to the size of the target molecule.
  • Prieto-Astalan et al. have also used magnetic particles to study specific binding of prostate specific antigen to the surfaces of the bioparticles comprised of clusters of magnetic single domains of magnetite, which are coated with dextrin (Prieto-Astalan, A. P et al. Biosensors and Bioelectrononics 2004, 19, 945-951; US 2003/9169032; US 2003/0076087; and WO 03/019188). Both groups showed that at sufficiently large amplitude of the magnetic field, or when using a dc-bias to the ac-excitation field, one observes a non-linear magnetic response. However, both of these approaches suffer from a number of limitations, e.g. low sensitivity.
  • a sensor for detecting an analyte comprises
  • the protein rod body portion comprises a tail fiber protein from a T even bacteriophage or a derivative thereof, for example a tail fiber protein of T4 bacteriophage such as a gp 34, gp35, gp36 or gp 37 tail fiber protein or a derivative thereof.
  • the protein rod body portion is a fusion protein comprising portions of two or more tail fiber proteins and/or a multimer of protein rod body portions, each having a magnetic nanoparticle and an analyte binding moiety associated therewith.
  • the present invention also provides a method for detecting the presence of an analyte in a liquid sample comprising the steps of: (a) placing a sensor in accordance with the invention into the liquid sample,
  • FIG. IA depict a schematic representation of the T4 bacteriophage particle (FIG. IA), and a schematic representation of the T4 bacteriophage tail-fiber (FIG. IB).
  • FIG. 2 A and B depicts a schematic representation of an electronic detection system.
  • An amplitude and frequency-variable ac magnetic field is provided by the primary solenoid 201 wound outside two series-opposing secondary coils 202, 203. These are wound directly on a capillary 204 for close coupling to the solution within 205.
  • the output voltage of the pair of coils is null, unless media with unequal magnetic characteristics are introduced in the two secondaries, at which point the output voltage, detected by a phase-sensitive detector, is non-zero.
  • the overall size of the system can be very small; secondaries wound with #50- wire are common.
  • the primary coil which is the source of the ac magnetic field, carries milli-ampere currents so is wound with a longer pitch and a somewhat more robust wire.
  • the phase sensitive detector allows measurement of both the in-phase (real) component of the magnetic susceptibility, ⁇ ' ( ⁇ ), and the out-of -phase (imaginary) component, ⁇ " ( ⁇ ).
  • Key components are the ac current generator, null electronic circuitry for balancing secondaries in absence of unequal media (N), phase sensitive detector ( ⁇ ), ⁇ is the sample capillary with media exposed to target protein, virus, cell or other molecule, and ⁇ is the sample capillary with media unexposed.
  • Figures 3 A-D depict attachment to a phage by a monoclonal antibody.
  • A Treatment of phage with mAb and secondary anti-serum. Each phage type was treated with 1 ⁇ g of mAb as described in Example 7.
  • B Time course of mAb treatment. S ⁇ lras2 phage were treated with 3 ⁇ g of mAb for the indicated time before a 30 min incubation with secondary anti-serum.
  • C Dose-response study of S ⁇ lras2 phage with varying amounts of mAb.
  • D Effect of treating mAb with free epitope before inactivation of S ⁇ lras2.
  • Figure 4 depicts a schematic of a nanosensor.
  • the construct is an engineered viral protein 41 conjugated to a superparamagnetic nanoparticle 42.
  • the distal region of the rod-like protein displays peptide/protein ligands 43 that bind to targets in solution. Binding is detected via resultant changes in rotational diffusion time by measuring the loss term of the complex magnetic susceptibility.
  • the present invention related to molecular protein/magnetic particle composite sensors that have affinity for a selected target analyte.
  • the protein component of the sensor is a rigid, rod-like structure engineered from a T4 tail-fiber gene.
  • Predetermined loci for binding nano-functional moieties can be genetically engineered along the distal half of the T4 tail-fiber (a structure that is about 2 nm wide by about 70 nm long - i.e., a "strut").
  • strut a structure that is about 2 nm wide by about 70 nm long - i.e., a "strut"
  • target specific binding moieties can serve to capture targets (e.g. molecules, viruses or cells) in solution. See Hyman, P. et al. PNAS 2000, 99, 4888-4893.
  • a magnetic particle for example a nanoparticle with a diameter of about 10 nm
  • the sensor of the present invention provides designability and rigid aspect ratio.
  • ⁇ the imaginary component of the magnetic susceptibility
  • the Brownian motion of such rigid asymmetric sensors is a cubic function of the length of the strut.
  • sensors of different lengths and different target analyte specificity can be synthesized whose peak frequencies will be significantly separated in the spectrum and facilitate de-convolution.
  • nano-sensors i.e., nanometer-sized sensors useful in the detection of biological and chemical entities.
  • the basis for the sensors described herein is a multivalent "nano-strut" (i.e., a long and thin rigid rod) composed of T-even tail-fiber proteins and variants thereof.
  • the present invention pertains to a nano-strut engineered to bind a paramagnetic nanoparticle, or other strongly magnetic molecular entity, and a target entity.
  • Tail fiber proteins used in the sensors of the invention can be modified in various ways to form novel rod structures with different properties. Specific internal peptide sequences can be deleted without affecting their ability to form trimers and associate with their natural tail-fiber partners. Alternatively, they can be modified so that they contain additional functional groups which enable them to interact with heterologous binding moieties.
  • the present invention also encompasses fusion proteins that contain sequences from two or more different tail-fiber proteins.
  • the present invention provides nano-sensors comprising native and modified tail-fiber proteins of bacteriophage T4.
  • the surface display ligands (for example peptide display ligands "PDL”) of the strut are genetically engineered into sections (e.g., the display ligand will bind a superparamagnetic or paramagnetic nanoparticle, and at a separate remote location, for example at the other end of the protein rod, will be a display specific for the selected target/analyte).
  • the display ligand will bind a superparamagnetic or paramagnetic nanoparticle, and at a separate remote location, for example at the other end of the protein rod, will be a display specific for the selected target/analyte).
  • a nano-strut composed of T4 tail-fiber proteins is a homb-trimer, and therefore most likely has three-fold symmetry along the long axis. This implies that any display is repeated three times about the specific locus on the axis.
  • the strut When the strut is put into a solution containing target(s), e.g., in a capillary, the struts will orient to some degree and point in the long direction. This orientation can be improved by a homogeneous DC magnetic field. By imposing a magnetic field gradient, the struts increase their concentration and therefore their alignment. In an ac magnetic field, at certain resonance frequencies, the struts will respond by an oscillatory movement, either along the axis or at an angle to it.
  • struts with a bound target can be distinguished from those without bound targets and quantified. Alignment of the struts and their dynamics may be monitored by passage of polarized light perpendicular to the tube axis (when using a DC field).
  • polarized light perpendicular to the tube axis (when using a DC field).
  • nano-struts of different lengths each genetically engineered to attract a different target, we can simultaneously assay several targets. Since the resonance frequency and strength are a function of length, mass and shape, the targets can be recognized in the same test by scanning the proper frequencies. Importantly, the test can be confirmed by titration using first a specific free peptide competitor and then a second.
  • peptides are same sequence as the target recognition peptides engineered into the struts and function as competitive inhibitors. If added one at a time, they should diminish each resonance amplitude in a specific manner. For example, a target molecule with two fold symmetry may well have two target sites and therefore the inhibition curve will be different from that of a target molecule which has only one. In each case (at each resonance frequency) the final value will give the same value as the starting value (before the addition of any target molecules). In this way the specificity of the signal can be checked (based on previous control data) and the baseline of the electronics and optics checked for changes or errors in the hardware. Finally, this approach allows for automated as well as manual analysis for specific sets of targets in a single sample.
  • AC and ac refer to alternating current
  • DC and dc refer to direct current
  • Chimers are defined herein as chimeric proteins in which at least the amino- and carboxy-terminal regions are derived from different original polypeptides, whether the original polypeptides are naturally occurring or have been modified by mutagenesis.
  • the peptide portion of a chimer is denoted by a tilda, " ⁇ ", and can be designated, e.g., gp(37 ⁇ 36) for a monomer and P(37 ⁇ 36) for a trimer.
  • gp denotes a monomeric polypeptide
  • P denotes homooligomers
  • P34, P36, and P37 are homotrimers.
  • Homotrimers are defined herein as assemblies of three substantially identical protein subunits that form a defined three-dimensional structure.
  • An isolated polypeptide that "consists essentially of a specified amino acid sequence is defined herein as a polypeptide having the specified sequence or a polypeptide that contains conservative substitutions within that sequence.
  • Conservative substitutions are ones in which an acidic residue is replaced by an acidic residue, a basic residue by a basic residue, or a hydrophobic residue by a hydrophobic residue.
  • the prefix "nano” indicates a structure of small size measured in nanometers, but generally greater than 1000 nm (1 ⁇ m) in any dimension.
  • the size of the sensors of the invention is substantially dependent on the size of the analyte, since greater observable change in the properties of the sensor in the magnetic field will occur with a greater percentage size increase when the sensor analyte complex is formed.
  • smaller analytes are most effectively detected with smaller sensors, while larger analytes can be detected with larger sensors.
  • binding will be understood in the manner conventional in the art to refer to non-covalent interactions such as those formed between antigens and their cognate antibodies, bio-receptors and their cognate ligands and like pairings, as may be reflected in binding, displacement or competition assays.
  • the position of the analyte binding is “remote” from the magnetic particle.
  • the magnetic particle and the analyte binding moiety are disposed at or near opposing ends of the protein rod body portion to maximize the change in shape and size when a sensor/analyte complex is formed. This degree of separation is not required, however, and the term "remote" is intended to reflect merely that the analyte binding moiety and the location of the magnetic particle are sufficiently separated that the there is no interference in binding both analyte and magnetic particle.
  • ⁇ "( ⁇ ) ⁇ /tl+ ⁇ 2 ] [Eq.l]
  • X 0 the inherent susceptibility
  • ⁇ the frequency
  • ⁇ the relaxation time.
  • ⁇ the relaxation time.
  • ⁇ r, rod ⁇ L 3 /3kT(ln(L/d - 0.8)) [Eq.3] where ⁇ is the fluid viscosity, r the spherical particle radius, L the rod length, d the rod diameter, k is Boltzmann's constant and T the absolute temperature.
  • FIG. 4 One embodiment of our sensor ( Figure 4) is based on the distal tail-fiber of bacteriophage T4.
  • This is a rigid rod-like trimeric protein 41 approximately 2.5 x 50 nm.
  • the sensor is created by the conjugation of a superparamagnetic nanoparticle 42 at one end of the protein 41 and the engineering of a peptide/protein based binding motif (s) 43 at another point(s) along the rod.
  • the binding motifs 43 bind to analyte 44.
  • Avidin/biotin conjugation is anticipated to provide magnetic nanoparticle attachment (N-terminal covalent attachment chemistry and phage display based discovery of peptides for direct nanoparticle binding are considered alternatives).
  • display peptides on the surface of the tail-fiber that bind antibody.
  • Assay of anthrax infection can be done using a high affinity display peptide specific to Bacillus anthracis protective antigen protein (PA).
  • PA Bacillus anthracis protective antigen protein
  • PA Bacillus anthracis protective antigen protein
  • PA Bacillus anthracis protective antigen protein
  • PA is the central soluble component of the tripartite anthrax toxin in blood and is most often used as an index of toxin production.
  • affinity display we can detect anthrax spores. (Collier, RJ.; Young, J.A.T. "Anthrax toxin” Ann Rev Cell & Dev Biol 2003, 19, 45-70.)
  • HYDRO Garcia de Ia Torre, et al, Biophysical Journal 1994, 67: 530-531.
  • HYDRO is based on the work of Bloomfield and colleagues (see Carrasco, et al, Biophysical Journal 1999, 75: 3044-3057 for a review) and uses bead modeling to approximate the shape of complex macromolecules.
  • the T4 37 ⁇ 1 distal half fibers (DHFs) useful in sensors of the invention have dimensions approximately 50nm x 2.5nm and were modeled as 20 beads with a diameter of 2.5nm.
  • the Mag/Rod ⁇ sensor was modeled as the DHF with a 5nm magnetic particle appended to the end of the rod.
  • Tail Fiber ⁇ r (s) % Change ⁇ l/ ⁇ r (kHz)
  • T4 tail-fiber proteins are also applicable to tail-fiber proteins of other T-even-like phage, such as the tail-fiber proteins of the T4 (e.g., T4, Tula, TuIb, etc.) or the T2 (e.g., T2, T6, K3, Ox2, Ml, etc.) families.
  • T4 e.g., T4, Tula, TuIb, etc.
  • T2 e.g., T2, T6, K3, Ox2, Ml, etc.
  • Bacteriophage (phage) T4 is one of the archetypal members of the family Myoviridae or T-even-like phage. These viruses are characterized by a large, elongated icosohedral head (which contains the phage DNA), a contractile tail sheath (and a "morphing" baseplate to stabilize the phage perpendicular to the cell, and to penetrate the outer cell wall preparatory to DNA injection), and tail-fibers (which contain the reversible receptors phage of the host receptors and trigger infection) (Wood, W. B. (1979) Harvey Lect. 73, 203-223; and Eiserling, F. A. & Black, L. W.
  • the tail-fiber proteins have an unusual quaternary structure of long, thin (3 nm x 150 nm), rigid rods (Beckendorf, S. K. J. MoI. Biol. 1973, 73, 37-53). Their function is to transduce chemical recognition of the bacteria host into a mechanical force on the phage base plate, essentially acting as a set of cooperative levers. This mechanical stress triggers a series of protein conformational changes that lead to entry of the phage DNA into the cell (Arscott, P. G.; Goldberg, E. B. Virology 1976, 69, 15-22; and Crawford, J. T.; Goldberg, E. B. J. MoI. Biol. 1980, 139, 679-690).
  • the three main tail-fiber proteins, P34, P36 and P37 (Note: gpX (gene product) refers to the monomeric product of gene X, whereas PX refers to the matured, multimeric complex of gpXs that has assembled into the structure that is found in the phage T4 virion) are principally composed of trimeric parallel "-helical rods (Eamshaw, W. C; Goldberg, E. B.; Crowther, R. A. J. MoL Biol. 1979, 132, 101-131). The monomer, gp35, that forms the angle in the tail-fiber, probably has a more complex structure.
  • the joints between the homotrimeric segments are also likely to have a more complex structure, but there is no evidence that the central rod regions have any tertiary structure (i.e., interactions between distant amino acid residues; Beckendorf, S. K. J. MoI. Biol. 1973, 73, 37-53).
  • the extended parallel ⁇ -helical secondary structure should directly support the rigid rod quaternary structure.
  • deletions or additions to the central rod regions which maintain the ⁇ -helical structure should permit alteration in tail-fiber length without greatly affecting overall structural integrity.
  • the binding domains at the ends of the proteins should form separate functional domains from the central, rigid rod domain.
  • the ⁇ -helical structure should contain turns and loops that can be expanded with functional peptides without disrupting the quaternary structure.
  • the present invention pertains to a class of protein building blocks whose dimensions are measured in nanometers, which are useful in the construction of sensors. It is believed that the basic unit of these "building blocks" is a homotrimer composed of three identical protein subunits having a helical-" configuration, although other oligomeric structures are possible. Thus, as will be apparent, references to a "homotrimer” or “trimerization” as used herein will in many instances be construed as also referring to other oligomers or oligomerizations. These long, stiff, and stable rod-shaped units can assemble with other rods using coupling devices that can be attached genetically or in vitro. The ends of one rod may attach to different ends of other rods or similar rods.
  • Variations in the length of the rods, in the angles of attachment, and in their flexibility characteristics permit differently-shaped structures to self-assemble in situ. In this manner, the units can self-assemble into predetermined larger structures.
  • the self-assembly can be staged to form structures of precise dimensions and uniform strength due to the relatively flawless biological manufacture of the components.
  • the rods can also be modified by genetic and chemical modifications to form predetermined specific attachment sites for other chemical and biological entities.
  • the protein units can be designed so that they comprise rods of different lengths, and can be further modified to include features that alter their surface properties in predetermined ways and/or influence their ability to join with other identical or different units.
  • the self-assembly capabilities can be expanded by producing chimeric proteins that combine the properties of two different members of this class. This design feature may be achieved by manipulating the structure of the genes encoding these proteins.
  • compositions and methods of the present invention take advantage of the properties of the natural proteins, i.e., the resulting structures are stiff, strong, stable in aqueous media, heat resistant, protease resistant, and can be rendered biodegradable. A large quantity of units can be fabricated easily in microorganisms. Further, for ease of automation, large quantities of parts and subassemblies can be stored and used as needed.
  • the sequences of the protein subunits of the instant invention are based on the components of the tail-fiber of the T4 bacteriophage of E. coli. It will be understood that the principles and techniques can be applied to the tail-fibers of other T-even-like phages, or other related bacteriophages that have similar tail and/or fiber structures.
  • P34, P36, and P37 are all stiff, rod-shaped protein homotrimers in which three identical " ⁇ -sheets, oriented in the same direction, are fused face-to-face, by hydrophobic interactions between, juxtaposed with a 120° rotational axis of symmetry through the long axis of the rod.
  • Gp35 is a monomeric polypeptide that attaches specifically to the N-terminus of P36 and then to the C-terminus of P34 and forms an angle joint between two rods.
  • the genes encoding these proteins may be modified so as to make rods of different lengths with different combinations of ends.
  • the helix properties of the native proteins are particularly advantageous in this regard.
  • the ⁇ -helix is composed of parallel triangular groups of ⁇ -strands thought to form a prism shape with ⁇ -bends or loops at the three long edges of the prism.
  • the amino acid side chains of the strands alternate up and down out of the plane of the layer formed by the three homotrimeric strands.
  • the first property allows loops to be extended to form symmetric and specific attachment sites between the L and R surfaces, as well as to form attachment sites for other structures.
  • the core sections of the ⁇ -sheet can be shortened or lengthened by genetic manipulations, e.g., by splicing DNA regions encoding " ⁇ -loops, on the same edge of the sheet, to form new loops that exclude intervening peptides, or by inserting segments of peptide in an analogous manner by splicing at bend angles.
  • the second property allows amino acid side chains extending above and below the surface of the " ⁇ -sheet to be modified by genetic substitution or chemical coupling. Importantly, all of the above modifications are achieved without compromising the structural integrity of the rod. It will be understood by one skilled in the art that these properties allow a great deal of flexibility in designing units that can assemble into a broad variety of structures, some of which are detailed below.
  • the rods of the present invention function like "struts", e.g., wooden dowels, 2x4 studs or steel beams used in construction.
  • the surfaces are exactly reproducible at the molecular level and thereby fitted for specific attachments to similar or different units rods at fixed joining sites.
  • the surfaces are also modified to be more or less hydrophilic, including positively or negatively charged groups, and have protrusions built in for specific binding to other units or to an intermediate joint with two receptor sites.
  • the three dimensions of the rod are defined as: x, for the back (B) to front (F) dimension; y, for the down (D) to up (U) dimension; and z, for the left (L) to right (R) dimension.
  • One dimensional multi-unit rods can be most readily assembled from single unit rods joined along the x axis but regular joining of subunits in either of the other two dimensions will also form a long structure, but with different cross sections than in the x dimension.
  • the protein subunits that are used to construct the nanosensors of the present invention are based on the four polypeptides that comprise the tail-fibers of bacteriophage T4, i.e., gp34, gp35, gp36 and gp37.
  • the genes encoding these proteins have been cloned, and their DNA and protein sequences have been determined (for gene 36 and 37 see Oliver et al. J. MoI. Biol. 1981, 153, 545-568).
  • the DNA and amino acid sequences of genes 34, 35, 36 and 37 are set forth in US Patent 5,877,279, which is hereby incorporated by reference in its entirety.
  • Gp34, gp35, gp36, and gp37 are produced naturally following infection of E. coli cells by intact T4 phage particles. Following synthesis in the cytoplasm of the bacterial cell, the g ⁇ 34, 36, and 37 monomers form homotrimers, which are competent for assembly into maturing phage particles.
  • E. coli serves as an efficient and convenient factory for synthesis and trimerization of the protein subunits described herein below.
  • the genes encoding the proteins of interest are incorporated into DNA expression vectors that are well known in the art, as discussed below.
  • circular plasmids typically contain selectable marker genes (usually conferring antibiotic resistance to transformed bacteria), sequences that allow replication of the plasmid to high copy number in E. coli, and a multiple cloning site immediately downstream of an inducible promoter and ribosome binding site.
  • selectable marker genes usually conferring antibiotic resistance to transformed bacteria
  • sequences that allow replication of the plasmid to high copy number in E. coli and a multiple cloning site immediately downstream of an inducible promoter and ribosome binding site.
  • Examples of commercially available vectors suitable for use in the present invention include the pET system (Novagen, Inc., Madison, Wis.) and Superlinker vectors pSE280 and pSE380 (Invitrogen, San Diego, Calif.).
  • the protocol is to 1) construct the gene of interest and clone it into the multiple cloning site; 2) transform E. coli cells with the recombinant plasmid; 3) induce the expression of the cloned gene; 4) test for synthesis of the protein product; and 5) test for the formation of functional homotrimers.
  • additional genes are also cloned into the same plasmid, when their function is required for trimerization of the protein of interest.
  • the bacterial chaperon gene 57 may also be included; when wild-type or modified gp36 is expressed, the wild-type version or a modified version of the gp37 gene may be included.
  • the modified gp37 should have the capacity to trimerize and contain an N-terminus that can chaperon the trimerization of gp36. This method allows the formation of monomeric gene products and, in some cases, maturation of monomers to homotrimeric rods in the absence of other phage-induced proteins normally present in a T4-infected cell.
  • Steps 1-4 of the aforementioned protocol may be achieved by methods that are well known in the art of recombinant DNA technology and protein expression in bacteria.
  • a representative of this type of chimer, the fusion of gp37-36, is described in Example 2.
  • the preferred hosts for production of these proteins are E. coli strain BL21(DE3) and BL21(DE3/pLysS) (available commercially from Novagen, Madison, Wis.), although other compatible recA strains, such as HMS174(DE3) and HMS174(DE3/pLysS) can be used.
  • Transformation with the recombinant plasmid may be accomplished by standard methods (Sambrook, J., Molecular cloning, Cold Spring Harbor Laboratories, Cold Spring Harbor, N. Y.; this monograph is also the source for many standard recombinant DNA methods used in this invention.)
  • Transformed bacteria may be selected by virtue of their resistance to antibiotics, e.g., ampicillin or kanamycin.
  • the method by which expression of the cloned tail-fiber genes is induced depends upon the particular promoter used.
  • a preferred promoter is plac (with a lac I q on the vector to reduce background expression), which can be regulated by the addition of isopropylthiogalactoside (IPTG).
  • a second preferred promoter is pT7#10, which is specific to T7 RNA polymerase and is not recognized by E. coli RNA polymerase.
  • T7 RNA polymerase which is resistant to rifamycin, is encoded on the defective lambda DE lysogen in the E. coli BL21 chromosome.
  • T7 polymerase in BL21 (DE3) is super-repressed by the laciq gene in the plasmid and is induced and regulated by IPTG.
  • a culture of transformed bacteria is incubated with the inducer for a period of hours, during which the synthesis of the protein of interest is monitored.
  • extracts of the bacterial cells are prepared, and the T4 tail-fiber proteins are detected, for example, by SDS-polyacrylamide gel electrophoresis.
  • the modified protein is detected in bacterial extracts, it is usually necessary to ascertain whether or not it forms appropriate homotrimers (Step 4). This may be accomplished initially by testing whether the protein is recognized by an antiserum specific to the mature trimerized form of the protein. Resistance to protease degradation is also a useful assay for native structure (Granboulan, P, J. Gen. Micro.1983, 129, 2217-2228).
  • Tail-fiber-specific antisera may be prepared as described (Edgar, R. S; Lielausis, I. Genetics 1965, 52, 1187-1200; and Ward, S. et al. J. MoI. Biol. 1970, 54, 15-31). Briefly, whole T4 phage may be used as an immunogen; optionally, the resulting antiserum is then adsorbed with tail-less phage particles, thus removing all antibodies except those directed against the tail-fiber proteins. In a subsequent step, different aliquots of the antiserum may be adsorbed individually with extracts that each lack a particular tail-fiber protein.
  • an extract containing only tail-fiber components P34, gp35, and gp36 (derived from a cell infected with a mutant T4 lacking a functional gp37 gene) is used for absorption, the resulting antiserum will recognize only mature P37 and trimerized P36-P37.
  • a similar approach may be used to prepare individual antisera that recognize only mature (i.e., homotrimerized) P34 and P36 by adsorbing with extracts containing distal half tail-fibers or P34, gp35 and P37, respectively, and gp monomers of all.
  • An alternative is to raise antibody against purified tail-fiber halves, e.g., P34 and gp35-P36-P37.
  • Anti gp35-P36-P37 can then be adsorbed with P36-P37 to produce anti-gp35, and anti-P36 can be produced by adsorption with P37 and gp35.
  • Anti-P37, anti-gp35, and anti-P34 can also be produced directly by using purified P37, gp35, and P34 as immunogens. Another approach is to raise specific monoclonal antibodies against the different tail-fiber components or segments thereof.
  • Specific antibodies to subunits or tail parts may be used in any of the following ways to detect appropriately homotrimerized tail-fiber proteins: 1) Bacterial colonies may be screened for those expressing mature tail-fiber proteins by directly transferring the colonies, or samples of lysed or unlysed cultures, to nitrocellulose filters, lysing the bacterial cells on the filter if necessary, and incubating with specific antibodies. Formation of immune complexes may then be detected by methods widely used in the art (e.g., secondary antibody conjugated to a chromogenic enzyme or radiolabeled Staphylococcal Protein A.). This method is particularly useful to screen large numbers of colonies e.g., those produced by EXO-SIZE deletion as described above.
  • Bacterial cells expressing the protein of interest may be first metabolically labeled with 35 S-methionine, followed by preparation of extracts and incubation with the antiserum. The immune complexes may then be recovered by incubation with immobilized Protein A followed by centrifugation, after which they may be resolved by SDS-polyacrylamide gel electrophoresis.
  • the chimers of the invention comprise at least about the first 50 (N-terminal) amino acids of a first tail-fiber protein fused via a peptide bond to at least about the last 50 (C-terminal) amino acids of a second tail-fiber protein.
  • the first and second tail-fiber proteins can be the same or different proteins.
  • the chimers comprise an amino acid portion in the range of the first 10-60 amino acids from a tail-fiber protein fused to an amino acid portion in the range of the last 10-60 amino acids from a second tail-fiber protein.
  • each amino acid portion is at least 20 amino acids of the tail-fiber protein.
  • the chimers comprise portions, i.e., not full-length tail-fiber proteins, fused to one another.
  • the first tail-fiber protein portion of the chimer is from gp37, and the second tail-fiber protein portion is from gp36.
  • a chimer gp37-36 chimer
  • a gp36-34 chimer after oligomerization to form P36-34, can bind to gp35, and this unit can then polymerize.
  • the first portion is from gp37, and the second portion is from gp34.
  • the chimers of the invention are made by insertions or deletions within a ⁇ turn of the ⁇ structure of the tail-fiber proteins.
  • insertions into a tail-fiber sequence, or fusing to another tail-fiber protein sequence, is done so that sequences in ⁇ turns on the same edge of the ⁇ -sheet are joined.
  • nanostructures of the invention can also comprise tail-fiber protein deletion constructs that are truncated at one end, e.g., are lacking an amino- or carboxy- end (of at least 5 or 10 amino acids) of the molecule.
  • Such molecules truncated at the amino-terminus e.g., of truncated gp37, gp34, or gp36, can be used to "cap" a nanostructure, since, once incorporated, they will terminate polymerization.
  • Such molecules preferably comprise a fragment of a tail-fiber protein lacking at least the first 10, 20, or 60 amino terminal amino acids.
  • portions of the same or different tail-fiber proteins can be inserted into a tail-fiber chimer to lengthen the rod, or be deleted from a chimer, to shorten the rod.
  • native (non-recombinant) gp35 is isolated for use by growing up a bacteriophage T4 having an amber mutation in gene 36, in a su° bacterial strain (not an amber suppressor), and isolating gp35 from the resulting culture by standard methods.
  • P34, P36-P37, P37, and chimers derived from them are purified from E. coli cultures as mature trimers.
  • Gp35 and variants thereof are purified as monomers. Purification may be achieved by the following procedures or combinations thereof, using standard methods:
  • the C-terminal domain of P37 binds to the lipopolysaccharide of E. coli B.
  • Other T4-like phages have P37 analogues that bind other cell surface components such as OmpF or TSX protein.
  • the proteins have been engineered to include heterologous domains that act as ligands or binding sites, the cognate partner is immobilized on a solid matrix and used in affinity purification.
  • a heterologous domain can be biotin, which binds to a streptavidin-coated solid phase.
  • several components are co-expressed in the same bacterial cells, and subassemblies of larger nanostructures are purified subsequent to limited in vivo assembly, using one or more of the methods enumerated above.
  • the purified components may then be combined in vitro under conditions where assembly of the desired nanostructure occurs at temperatures between about 4 °C and about 37 °C, and at pHs between about 5 and about 9.
  • optimal conditions for assembly i.e., type and concentration of salts and metal ions
  • one or more crude bacterial extracts may be prepared, mixed, and assembly reactions allowed to proceed prior to purification.
  • one or more purified components assemble spontaneously into the desired structure, without the necessity for initiators.
  • an initiator is required to nucleate the polymerization of rods or sheets. This offers the advantage of localizing the assembly process (i.e., if the initiator is immobilized or otherwise localized) and of regulating the dimensions of the final structure.
  • rod components that contain a functional P36 C-terminus require a functional P37 N-terminus to initiate rod formation stoichiometrically; thus, altering the relative amount of initiator and rod component will influence the average length of polydisperse rod polymer.
  • the final nanostructure is composed of two or more components that cannot self-assemble individually, but only in combination with each other. In this situation, alternating cycles of assembly can be staged to produce final products of precisely defined structure (see Example 6B below).
  • an immobilized initiator When an immobilized initiator is used, it may be desirable to remove the polymerized unit from the matrix after staged assembly.
  • specialized initiators may be engineered so that the interaction with the first rod component is rendered reversibly thermolabile (see Example 5).
  • the polymer can be easily separated from the matrix-bound initiator, thereby permitting: 1) easy preparation of stock solutions of uniform parts or subassemblies, and 2) re-use of the matrix-bound initiator for additional cycles of polymer initiation, growth, and release.
  • the nanostructure may be made detachable using a mutant (thermolabile) gp34 that can be made to detach upon exposure to a higher temperature (e.g., 40 °C).
  • a mutant (thermolabile) gp34 that can be made to detach upon exposure to a higher temperature (e.g., 40 °C).
  • Such a mutant g ⁇ 34 termed T4 tsB45, having a mutation near its C-terminal end such that P34 attaches to the distal tail-fiber half at 30 0 C, but can be separated from it in vitro by incubation at 40° C in the presence of 1% SDS (unlike wild-type T4 which are stable under these conditions), has been reported (Seed, 1980, Studies of the Bacteriophage T4 Proximal Half Tail-fiber, Ph.D. Thesis, California Institute of Technology). Proteins which catalyze the formation of correct (lowest energy) stable secondary (2°) structure of proteins are called chaperone proteins.
  • this stabilization is aided by tertiary structure, e.g., stabilization of "-sheets by their interaction in ⁇ -barrels or by interaction with alpha-helices.
  • chaperonins prevent intrachain or interchain interactions which would produce untoward metastable folding intermediates and prevent or delay proper folding.
  • accessory proteins gp57 and gp38, in the morphogenesis of T4 phage tail-fibers which are sometimes called chaperonins because they are essential for proper maturation of the protein oligomers but are not present in the final structures.
  • chaperonin systems e.g., groEL/ES
  • groEL/ES oligopeptide moieties of the gene product to prevent unwanted interactions with oligopeptide moieties elsewhere on the same polypeptide or another peptide. These would form metastable folding intermediates which retard or prevent proper folding of the polypeptide to its native (lower energy) state.
  • Gp57 probably in conjunction with a membrane protein(s), may have the role of juxtaposing and/or initiating the folding of 2 or 3 identical gp37 molecules.
  • the gp38 protein may stabilize this interaction until the aligned peptides then zip up (while mutually stabilizing their nascent b-structures) to form a rod without further interaction with gp57 (Qu, et al., J. Bact, 2004, 186, 8363-8369).
  • Gp57 acts in T4 assembly not only for oligomerization of gp37 but also for gp34 and gpl2.
  • molecules that can self-assemble can be produced as fusion proteins, fused to the N- or C- terminus of tail-fiber variants of the invention (chimers, deletion/insertion constructs) to align their ends and thus to facilitate their subsequent unaided folding into oligomeric, stable ⁇ -helical rod-like (rod) units in vitro, in the absence of the normally required chaperonin proteins (e.g., gp57) and host cell membrane proteins.
  • the P37 unit as an initiator of gp(37 ⁇ 36) oligomerization and polymerization.
  • proper folding of gp37 to a P37 initiator requires a phage infected cell membrane, and two chaperone proteins, gp38 and gp57.
  • the need for gp38 can be obviated by use of a mutation, ts3813 (a duplication of 7 residues just downstream of the transition zone of gp37) which suppresses gene 38 (Wood, W. B., F. A. Eiserling and R A.
  • a moiety that self-assembles into a dimer or trimer or other oligomer (“self-assembling moiety”) is fused to a C-terminal deletion of gp37 downstream or upstream of the transition region [the transition region is a conserved 17 amino acid residue region in T4-like tail-fiber proteins where the structure of the protein narrows to a thin fiber; see Henning et al., 1994, "Receptor recognition by T-even-type coliphages," in Molecular Biology of Bacteriophage T4, Karam (ed.), American Society for Microbiology, Washington, D.C., pp.
  • the self-assembling moiety can be a self trimerizing mutant leucine zipper peptide, p ⁇ in which both the a and d positions are substituted with isoleucine (Harbury P. B., et al. ibid.).
  • a collagen peptide can be used as the self-assembling moiety, such as that described by Bella et al. (Bella, J.; Eaton, M.; Brodsky, B.; Berman, H. M. Science 1994, 226, 75-81), which self aligns by an inserted specific non-repeating alanine residue near the center.
  • tail-fiber acts as a trigger to signal initiation of the tail sheath contraction process that precedes phage DNA injection.
  • the reversible, noncovalent binding of a number of tail-fiber distal ends to their specific receptor sites on the cell surface leads to a cooperative mechanical stress in the base plate. This stress triggers base plate expansion and initiates the tail sheath contraction, which extends the tail core through the cell wall (Crawford, J. T.; Goldberg, E. G. J. MoI. Biol. 1977, 111, 305-313; and Crowther, R. A. J. MoI. Biol. 1980, 7, 159-174).
  • the tail-fibers' critical function for phage viability provides a sensitive assay for rigidity in tail-fiber structure because any substantial loss of rigidity in the structure should impair the tail-fibers' triggering function.
  • a deletion can be formed in T4 P37 spanning amino acids A175-N544.
  • the important factor is that the interserted peptide display be presented so that it can interact with a magnetic particle or a target analyte to form the sensor or sensor/ analyte complex, as is the case when the PDL Is inserted in a ⁇ -loop of P37.
  • the loop regions contribute little to maintaining the H-bond network, nor to the van der Waals interaction in the hydrophobic layer within the rod (Branden, C. & Tooze, J. (1999) Introduction to Protein Structure (Garland, New York), 2nd Ed.; and Xu, G.; Wang, W.; Groves, J. T. ; Hecht, M. H. Proc. Natl. Acad. Sci. USA 2001, 98, 3652-3657).
  • loops can be more variable and flexible than other regions of the tail-fiber proteins, demonstrating that the junction of the S ⁇ 1 deletion is in a loop (rather than in a ⁇ -strand) of the rod portion of gene 37.
  • Surface loops in proteins can often be expanded to include additional peptide sequences with minimal effects on protein structure, function or stability (Regan, L. Curr. Opin. Struct. Biol. 1999, 9, 494-499).
  • the S ⁇ 1 junction is in a loop, we can insert additional sequences into the junction, expanding the loop, without disrupting the structural integrity of the tail-fiber.
  • tail-fiber derived proteins as mesoscale assembly units, one needs to attach specific functions to the assembled arrays of structural units. They may be attached before or after maturation of the final structure or at an intermediate step. The attachment may be covalent (e.g., disulfide bridges) or noncovalent (e.g., His tags). Incorporation of a peptide epitope may also be used to attach a functionality linked to the appropriate antibody.
  • each mature fiber contains more than one epitope in close proximity on each tail-fiber, it is likely that both binding sites in the mAb become bound to a single fiber. This would not be expected to inactivate the phage.
  • Fig. 3B shows that inactivation depends on the time allowed for mAb binding before addition of the secondary antiserum, reaching a maximum of 99.9% by 120 min.
  • Fig. 3C shows that inactivation also has a simple dose-response relationship with the amount of Y13-259 mAb used.
  • Fig. 3D shows that the S ⁇ lras2 phage could be protected from inactivation by pretreating the mAb with a free 15-aa peptide of the same sequence as the 15-residue epitope inserted into the tail-fiber protein.
  • the length of the chimeric proteins can be adjusted by using more or less of the rod region from either of the parent proteins, allowing the spacing of the functional moieties to be controlled.
  • other ⁇ -loops within the central rod domain can be used as insertion sites for the addition of antigenic peptides that can subsequently be recognized by antibodies to add either functional or structural capabilities including crosslinking of the polymeric fibers into open two- and three-dimensional arrays.
  • Magnetic spherical particles with a diameter of less than about 10 nm are magnetic mono domains both in a magnetic field and in the zero field.
  • a particle being a magnetic mono domain means that the particle only contains one magnetization direction.
  • they can either be thermal blocked or super paramagnetic.
  • the direction of the magnetization for thermal blocked particles are oriented in a manner in the magnetic particle in proportion to the crystallographic orientation of the particle, and "locked" to this direction, meanwhile studying the particle system. Under the influence of an external magnetic field, the entire particle physically rotates so that its magnetization directions gradually coincide to some extent with the direction of the external added field.
  • Small magnetic particles can be manufactured from a variety of of materials, for example Fe 3 O 4 , Fe 2 O 4 , Fe 2 O 3 , cobalt-doped iron oxide or cobalt iron oxide (CoFe 2 O 4 ).
  • Other magnetic materials specifically (but not exclusively) rare earth metals (for example ytterbium or neodymium), their alloys or compounds containing rare earth metals, or doped magnetic (element) substances are also possible.
  • the sizes of the particles can be produced from about 3 nm to about 30 nm. The final size in this process depends on a number of different parameters during the manufacturing.
  • Magnetic nano-particles offer some attractive possibilities as components of nanosensors. First, they have controllable sizes ranging from a few nanometers up to tens of nanometers, which places them at dimensions that are smaller than or comparable to those of a cell (10-100 ⁇ m), a virus (20 ⁇ 50 nm), a protein (5-50 nm) or a gene (2 nm wide and 10-100 nm long). This means that they can 'get close' to a biological entity of interest. Indeed, they can be coated with biological molecules to make them interact with or bind to a biological entity, thereby providing a controllable means of 'tagging' or addressing it.
  • the nano-particles are magnetic and can be manipulated by an external magnetic field gradient.
  • This 'action at a distance' combined with the intrinsic penetrability of magnetic fields into human tissue, opens up many applications involving the transport and/or immobilization of magnetic nano-particles, or of magnetically tagged biological entities. In this way, they can be made to deliver a package, such as an anticancer drug, or a cohort of radionuclide atoms, to a targeted region of the body, such as a tumor.
  • the magnetic nano-particles can be made to respond resonantly to a time-varying magnetic field, with advantageous results related to the transfer of energy from the exciting field to the nanoparticle.
  • the particle is metallic, it can be made to heat up, which leads to their use as hyperthermia agents, delivering toxic amounts of thermal energy to targeted bodies such as tumors; or as chemotherapy and radiotherapy enhancement agents, where a moderate degree of tissue warming results in more effective malignant-cell destruction.
  • hyperthermia agents delivering toxic amounts of thermal energy to targeted bodies such as tumors
  • radiotherapy enhancement agents where a moderate degree of tissue warming results in more effective malignant-cell destruction.
  • the magnet (reporter) component of the nano-sensor is a magnetic nanoparticle.
  • a wide array of mono-disperse nanoparticles, with controlled size and magnetics, can be attached to the nano-sensors by specific high affinity PDLs to bind to the magnet and engineer the peptide to extend from termini of the high melting trimeric tetraheptad coiled-coil (Qu, Y. et al. J. Bacteriol. 2004, 186, 8363-8369).
  • the nano-magnets are paramagnetic nanoparticles comprised of one or more metals. In one embodiment, the nano-magnets are paramagnetic nanoparticles comprised of one or more transition metals. In a preferred embodiment, the nano-magnets are selected from paramagnetic nanoparticles consisting of one or more of the following metals or metalloids: cobalt, iron, gold, chromium, palladium, platinum, manganese, neodymium, nickel, zirconium, copper, niobium, boron and their oxides and alloys.
  • the paramagnetic nanoparticles are iron-based metals and alloys, and their oxides and nitrides (e.g., iron, iron-gold, iron-chromium, iron nitride (Fe-N), iron oxide (Fe3O4), iron-palladium, iron-platinum, and iron-neodymium-boron, iron-neodymium-boron-niobium-copper, ironzirconium-neodymium-boron.)
  • the nano-magnet is an transition-metal-containing biological entity.
  • the nano-magnet is an iron-containing biological entity (e.g. ferritin).
  • the nano-magnets are monodisperse MFe 2 O 4 , wherein M is iron, cobalt or manganese.
  • the magnetic nanoparticle has a diameter between about 0.1 nm and 100 nm. In a preferred embodiment, the magnetic nanoparticle has a diameter between about 1 nm and about 50 nm. In a preferred embodiment, the magnetic nanoparticle has a diameter between about 7 nm and about 15 nm. In a preferred embodiment, the magnetic nanoparticle has a diameter of about 11 nm.
  • Nanoparticles may be coated with a material to aid in their interaction with the nanostrut.
  • the magnetic nanoparticle is coated with a polymer.
  • the nanoparticle is coated with a biological polymeric material.
  • the magnetic nanoparticle is coated with nucleic acids, or oligosaccharides, or proteins.
  • the nano-sensors contain target recognition peptides specific to a biological molecule or chemical entity.
  • a "biological molecule” is broadly defined as a molecule which has been constructed from the compounds from which organisms are formed. Such compounds can be amino acids, nucleic acids, saccharides, membrane lipids, or biological cofactors.
  • Amino acids include the twenty essential amino acids and other amino acids which can be incorporated into proteins.
  • Molecules constructed from amino acids also include peptides, i.e., chains composed of amino acids linked together through peptide bonds. These can be molecules such as neurotransmitters, hormones, and/or peptides derived from the functional parts of larger peptides.
  • Molecules constructed from amino acids also include proteins, i.e., longer chained peptides which may have one of many noncatalytic functions, such as electron transfer proteins (e.g., ferredoxins and flavodoxins), immune protection proteins (antibodies), proteins that generate or transmit nerve impulses (e.g., acetylcholine, dopamine, and the rhodopsin membrane receptors), structural proteins (e.g., collagen, fibrin, glycoproteins, elastin, etc.), other binding proteins (e.g., histones), and mass transport proteins (e.g., ferritin, hemoglobin).
  • electron transfer proteins e.g., ferredoxins and flavodoxins
  • immune protection proteins antibodies
  • proteins that generate or transmit nerve impulses e.g., acetylcholine, dopamine, and the rhodopsin membrane receptors
  • structural proteins e.g., collagen, fibrin, glycoproteins,
  • Saccharides include monosaccharides (e.g., glucose, fructose), oligosaccharides (e.g., sucrose, and raffinose), and polysaccharides (e.g., starch, and cellulose).
  • Membrane lipids include molecules such as phospholipids (e.g., lipid bilayer membranes and other fatty acids), glycolipids, cholesterol and its derivatives, and prostaglandin and its derivatives.
  • Nucleic acids include the five common nucleotides (adenine, guanine, cytosine, uracil, and thymine), oligonucleotides, and polynucleotides or nucleic acids such as DNA, m-RNA, and t-RNA.
  • Cofactors are biological molecules whose catalytic function may not be generated or specifically directed until associated with a polypeptide chain. Examples are riboflavin derivatives, porphyrins, thiamin pyrosphosphate and nicotinamide adenine dinucleotide. Since specific interactions usually occur in biological systems it is probable that the sensor can have a distinguished role within this area, for example analysis of biochemical markers for different diseases. Examples of molecules that can interact specifically with each other are: a) antibody-antigen; b) receptor-hormone; c) two complementary single strands of DNA; and d) enzyme-substrate/enzyme-inhibitor.
  • One aspect of the invention relates to detecting changes in the magnetic response of the magnetic particles that depend on the Brownian relaxation ( ⁇ ) in a carrier fluid (for example, water or a suitable buffer fluid, or another fluid suitable for the biomolecules that are the final target for the detection) under influence of an external AC-magnetic field.
  • a carrier fluid for example, water or a suitable buffer fluid, or another fluid suitable for the biomolecules that are the final target for the detection
  • the hydrodynamic volume of respective particles will be increased resulting in a decrease of the frequency, f max , wherein the out-of -phase component of the magnetic susceptibility is at its maximum.
  • the initially mentioned method further involves modification of the effective volume of the particle or its interaction with the carrier fluid as the hydrodynamic volume of the particle changes.
  • the measurement is actually a relative measurement because changes in a modified particle system are compared with an original system. Therefore, least two sample containers and two detector coils are used for the measurement.
  • the amplitude and phase of the voltage output from the secondary coil set is a measure of the magnetic susceptibility of the specimen to be analyzed.
  • An external oscillator/frequency generator can be arranged, such that the coils are in an bridge circuit.
  • the difference between the induced voltage output of both detector coils is measured, and, in addition, the phase difference between it and that of the frequency generator is measured.
  • a noise source can be used as well and the response of the system can be analyzed by means of a FFT (Fast Fourier Transform) analysis of an output signal.
  • the signal difference is set to zero between the secondary coils, which is done through adjustment of the passive resistance and inductive components of an external (nulling) circuit attached to one of the secondary coils.
  • the zero setting can be done through minimizing the signal through adding a determined amount of a magnetic sensors in one of the spaces wherein the sample containers are placed, so that the substance creates an extra contribution to the original signal that therefore can be set to zero.
  • Figures 2 A and B show a schematic representation of device capable of making this measurement.
  • the primary coil 201 and its associated circuitry are separated from the secondary coils 202 and 203, and the capillary 204 is not shown for clarity.
  • Fig. 2B shows cross section through secondary coil 202 depicting the relative positions of the primary coil 201, the secondary coil 202 and the capillary 204 having sample space 205.
  • an ac current is passed through coil 201 of the primary circuit, resulting in the generation of an oscillating magnetic field within the coil. The strength of this field depends on the magnitude of the signal.
  • the magnetic field results in an oscillation of sensor molecules within the sample chambers ⁇ and ⁇ of the capillary 204.
  • Sample chamber ⁇ is disposed within secondary coil 202 and sample chamber ⁇ coils is disposed within secondary coil 203.
  • Secondary coils 202 and 203 are in series, but are wound in opposite directions.
  • sample potentially containing analyte can be added to both sample chambers.
  • the two chambers are then titrated, one with a control solution and the other with the control solution containing a competitor for the binding of the sensor to the analyte. If analyte is present, this will result in sensor being freed on one side of the chamber as a result of competitive binding, but not on the other.
  • the resulting change in the magnetic properties of the two chambers can therefore be observed in the same manner, although the change will be in the opposite direction.
  • the fluid sample comprises one or several proteins in a fluid solution, like blood, blood plasma, serum, saliva, feces, urine or samples from water, waste, or soil (for environmental testing).
  • the analysis can be connected to the particle through interaction with a second molecule, which is connected to the particle before the analysis starts. Interactions that may be probed include antibody-antigen, receptor-hormone, two complementary single strings of DNA and enzyme-substrate/enzyme-inhibitor.
  • the invention also relates to a method for detection of changes in the magnetic response of at least one magnetic particle provided with an external layer in a carrier fluid, which method comprises measurements of the magnetic particles characteristic rotation period with respect to the agitation of the external layer.
  • the method uses at least two substantially identical detection coils connected to detection electronics and sample containers for absorbing carrier fluid.
  • An excitation coil can surround the detection coils and sample containers for generation of a homogeneous magnetic field at the sample container.
  • the excitation coil, measurement coils and sample container are placed concentric and adjusted round its vertical center axis.
  • the arrangement can furthermore comprise an oscillator system wherein the detection coils constitute the frequency-determining element in an oscillator circuit.
  • the coils are arranged in the oscillator return coil.
  • the coils that surround the samples respectively are electrically phase-shifted versus each other (series opposing), so that when properly balanced by the nulling circuit, the output of the system measured by a phase sensitive detector, is zero (both in- and out-of-phase components).
  • an operational amplifier can be arranged to subtract two voltages from each other. Consequently when two samples with unequal magnetic response are inserted in the two coils, the output signal is non-zero.
  • the arrangement comprises a phase-locking circuit in one embodiment.
  • the arrangement comprises oscillator/frequency generator signal to generate period variable current to excite the coils by means of white noise. Frequency depending information is received through an FFT-filtering of the response.
  • Nanosensors according to the invention have applications in many different areas.
  • An important area is that of clinical medicine, where numerous analytical instruments and techniques are currently employed to determine the concentration of clinically important markers.
  • radioimmunoassay techniques were used for most chemical analyses, but these require typically large and expensive instruments designed for large centralized hospital or clinical laboratories. Pressures to cut health-care costs are creating more demand for less expensive, smaller analyzers which can be used in decentralized organizations, e.g., in individual hospital wards, outpatient departments, and physicians' offices.
  • the nanosensors of the invention as a result of their small size, low cost, selectivity, and sensitivity, may fill that need.
  • Various chemical sensors exist for potassium, sodium, hydrogen, lithium, and calcium ions, but have yet to be developed for most proteins, hormones, metabolites, and organic drugs.
  • the senor can, for example, be used within medical diagnostics.
  • the biosensor could for example replace some ELISA analysis (Enzyme Linked Immunsorbent Assay). This method is used today to a great extent to determine contents of biochemical markers (for example proteins) found in complex body fluids, such as blood, serum and cerebro-spinal fluid.
  • ELISA analysis Enzyme Linked Immunsorbent Assay
  • ELISA analysis that can replace the new biosensor are: a) analysis of tau proteins in cerebro-spinal fluid (part of diagnosis of Alzheimer's disease); b) analysis of PSA in serum (diagnosis of prostate cancer); c) analysis of acute phase proteins measured in connection with heart disease; and d) analysis of CA 125 in serum (diagnosis of cancer in the ovaries).
  • Biosensors have many potential applications in the diagnosis and monitoring of animal health problems.
  • the defense industry also has a need for sensitive chemical sensors to monitor trace levels of chemical and biological warfare agents.
  • Other applications for chemical sensors include food and feed process and quality control, agricultural diagnostics and monitoring, industrial hygiene, and toxicology testing.
  • the sensors and methods of the present invention can be used for "low throughput screening", that is the accomplishment of one or several analysis at the same time, or for "high throughput screening", that is the accomplishment of a large number of analyses simultaneously.
  • the gene encoding gp37 contains two sites for the restriction enzyme BgI ⁇ , the first cleavage occurring after nucleotide 293 and the second after nucleotide 1486 (the nucleotides are numbered from the initiator methionine codon ATG.)
  • BgI ⁇ restriction enzyme
  • the gene encoding gp37 contains two sites for the restriction enzyme BgI ⁇ , the first cleavage occurring after nucleotide 293 and the second after nucleotide 1486 (the nucleotides are numbered from the initiator methionine codon ATG.)
  • the restriction digestion reaction mix contains: 2 /xL of gp37 plasmid DNA (1 ⁇ g/ ⁇ L), 1 ⁇ L NEB buffer #2 (10X), 6 ⁇ L H 2 O (1 ⁇ L), and 1 ⁇ L BgI II (10 U/ ⁇ L).
  • the gp37 plasmid signifies a pT7-5 plasmid into which gene 37 has been inserted in the multiple cloning site, downstream of a good ribosome binding site and of gene 57 to chaperone the trimerization.
  • the reaction is incubated for 1 h at 37° C.
  • 89 ⁇ L of T4 DNA ligase buffer and 1 ⁇ L of T4 DNA ligase are added, and the reaction is continued at 16° C for 4 hours.
  • the reaction mixture is then transformed into E. coli strain BL21, obtained from Novagen, using standard procedures.
  • the transformation mixture is plated onto nutrient agar containing 100 ⁇ g/ml ampicillin, and the plates are incubated overnight at 37° C.
  • Colonies that appear after overnight incubation are picked, and plasmid DNA is extracted and digested with BgI II as above.
  • the restriction digests are resolved on 1% agarose gels.
  • a successful deletion is evidenced by the appearance after gel electrophoresis of a new DNA fragment of 4.2 kbp, representing the undeleted part of gene 37 which is still attached to the plasmid and which re-formed a BgIII site by ligation.
  • the 1.2 kbp DNA fragment bounded by BgI II sites in the original gene is no longer in the plasmid and so is missing from the gel.
  • Plasmids selected for the predicted deletion as above are transformed into E. coli strain BL21(DE3). Transformants are grown at 30° C until the density (A600) of the culture reaches 0.6 IPTG is then added to a final concentration of 0.4 mM and incubation is continued at 30° C for 2 h, after which the cultures are chilled on ice. 20 ⁇ L of the culture is then removed and added to 20 ⁇ L of a two-fold concentrated "cracking buffer" containing 1% sodium dodecyl sulfate, glycerol, and tracking dye.
  • a two-fold concentrated "cracking buffer" containing 1% sodium dodecyl sulfate, glycerol, and tracking dye.
  • the ability of the deleted polypeptide to trimerize appropriately is directly evaluated by testing its ability to be recognized by an anti-P37 antiserum that reacts only with mature P37 trimers, using a standard protein immunoblotting procedure.
  • An alternative assay for functional trimerization of the deleted P37 polypeptide is its ability to complement in vivo a T4 37- phage, by first inducing expression of the ⁇ P37 and then infecting with the T4 mutant, and detecting progeny phage.
  • a ⁇ P37 was prepared as described above, and found capable of complementing a T4 37- phage in vivo.
  • EXAMPLE 2 Design, Construction and Expression of a gp37-36 Chimer
  • the starting plasmid for this construction is one in which the gene encoding gp37 is cloned immediately upstream (i.e., 5') of the gene encoding gp36.
  • the plasmid is digested with Hae m, which deletes the entire 3' region of gp37 DNA downstream of nucleotide 724 to the 3'-terminus, and also removes the 5' end of gp36 DNA from the 5' terminus to nucleotide 349.
  • the reaction mixture is identical to that described in Example 1, except that a different plasmid DNA is used, and the enzyme is HaeDI.
  • gp37-36 chimer Successful expression of the gp37-36 chimer is evidenced by the appearance of a protein product of about 35,000 daltons.
  • This protein will have the first 242 N-terminal amino acids of gp37 fused to the final 104 C-terminal amino acids of gp36 (numbered 118-221)
  • the utility of this chimer depends upon its ability to trimerize and attach end-to-end. That is, carboxy termini of said polypeptide will have the capability of interacting with the amino terminus of the P37 protein trimer of bacteriophage T4 and to form an attached diner, and the amino terminus of the trimer of said polypeptide will have the capability of interacting with other said chimer polypeptides. This property can be tested by assaying whether introduction of ⁇ P37 initiates trimerization and polymerization. Alternatively, polyclonal antibodies specific to P36 trimer may be used to detect P36 subsequent to initiation of trimerization by ⁇ P37.
  • a gp37-36 chimer was prepared similarly to the procedures described above, except that the restriction enzyme Taql was used instead of Haei ⁇ . Briefly, the 5' fragment resulting from Taql digestion of gene 37 was ligated to the 3' fragment resulting from Taql digestion of gene 36. This produced a construct encoding a gp37-36 chimer in which amino acids 1-48 of gp37 were fused to amino acids 100-221 of gp36. This construct was expressed in E. coli BL21 (DE3), and the chimer was detected as an 18 kD protein. This gp37-36 chimer was found to inhibit the growth of wild type T4 when expression of the gp37-36 chimer was induced prior to infection (in an in vitro phage inhibition assay).
  • the goal of this construction is to produce two variants of a trimerizable P37-36 chimer: One in which the N-terminus of the polypeptide is mutated (A, designated *P37-36) and one in which the C-terminus of the polypeptide is mutated (B, designated P37-36*).
  • A designated *P37-36
  • B designated P37-36*
  • the requirement is that the mutated *P37 N-terminus cannot form a joint with the wild-type P36 C-terminus, but only with the mutated *P36 N-terminus.
  • a and B each cannot polymerize independently (as the parent P37-36 protein can), but can only associate with each other sequentially (i.e., P37-36* + *P37-36 % P37-36*&*P37-36).
  • a second construct, *p37-P36* is formed by recombining *P37-36 and P37-36* in vitro.
  • the monomers *gp37-36* and gp37-36 are mixed in the presence of P37 initiator, gp37-36 would trimerize and polymerize to (P37-36)n; similarly, *P37 would only catalyze the polymerization of *gp37-36* to (*P37-36*)n.
  • the two chimers could be of different size and different primary sequence with different potential side-group interactions, and could initiate attachment at different surfaces depending on the attachment specificity of P37.
  • the starting bacterial strain is a su 0 strain of E. coli (which lacks the ability to suppress amber mutations).
  • This strain is infected with a mutant T4 bacteriophage containing amber mutations in genes 35, 36, and 37, phage replication is incomplete, since the tail-fiber proteins cannot be synthesized.
  • this strain is first transformed with a plasmid that directs the expression of the wild type gp35, gp36and gp37 genes and induced with IPTG, and subsequently infected with mutant phage, infectious phage particles are produced; this is evidenced by the appearance of "nibbled" colonies. Nibbled colonies do not appear round, with smooth edges, but rather have sectors missing. This is caused by attack of a microcolony by a single phage, which replicates and prevents the growth of the bacteria in the missing sector.
  • the 3 '-terminal region of gene 36 (corresponding to the C-terminal region of gp36) is mutagenized with randomly doped oligonucleotides.
  • Randomly doped oligonucleotides are prepared during chemical synthesis of oligonucleotides, by adding a trace amount (up to a few percent) of the other three nucleotides at a given position, so that the resulting oligonucleotide mix has a small percentage of incorrect nucleotides at that position. Incorporation of such oligonucleotides into the plasmid will result in random mutations (Hutchison, C. A. IH et al. Methods. Enzymol. 1991, 202, 356-390).
  • the mutagenized population of plasmids (containing, however, unmodified genes 36 and 37), is then transformed into the su° bacteria, followed by infection with the mutant T4 phage as above.
  • the appearance of non-"nibbled" colonies indicates that the mutated gp36 C-termini can no longer interact with wild type P37 to form functional tail-fibers.
  • the putative gp36* phenotypes found in such non-nibbled colonies are checked for lack of trimeric N-termini by appropriate immunospecificity as outlined above, and positive colonies are used as source of plasmid for the next step.
  • the *P37-36 and P37-36* paired suppressor chimers are then constructed in the same manner as described in Example 2. In this case, however, *P37 is used in place of wild type P37 and P36* is used in place of wild type P36.
  • a *P37-36* chimer can now be made by restriction of *P37-36 and P37-36* and religation in the recombined order.
  • the *P37-36* can be mixed with the P37-36 chimer, and the polymerization of each can be accomplished independently in the presence of the other. This is useful when the rod-like central portion of these chimers have been modified in different ways.
  • EXAMPLE 4 Design, Construction and Expression of a gp36-34 Chimer
  • the starting plasmid for this construction is one in which the vector containing gene 57 and the gene encoding gp36 is cloned immediately upstream (i.e., 5') of the gene encoding gp34.
  • the plasmid is digested with Ndel, which cuts after bp 219 of gene 36 and after bp 2594 of gene 34, thereby deleting the final 148 C-terminal codons from the pg36 moiety and the first 865 N-terminal codons from the gp34 moiety.
  • the reaction mixture is identical to that described in Example 1, except that a different plasmid DNA is used, and the enzyme used is Ndel (NEB).
  • the starting plasmid is cut with Sphl at bp 648 in gene 34, and the Exo-Size Deletion Kit (NEB) is used to create deletions as described above.
  • the resulting construct is then expressed in E. coli BL21(DE3) as described in Example 1.
  • Successful expression of the gp36-34 chimer is evidenced by the appearance of a protein product of about 55,000 daltons.
  • the amino termini of the polypeptide homotrimer have the capability of interacting with the gp35 protein, and then the carboxy termini have the capability of interacting with other attached gp35 molecules.
  • Successful formation of the trimer can be detected by reaction with anti-P36 antibodies or by attachment of gp35 or by the in vitro phage inhibition assay described in Example 2.
  • the P37-36 chimer described in Example 2 is capable of self-assembly, but requires a P37 initiator to bind the first unit of the rod. Therefore, a P37 or a ⁇ P37 trimer is either attached to a solid matrix or is free in solution to serve as an initiator. If the initiator is, attached to a solid matrix, a thermolabile P37 trimer is preferably used. Addition of an extract containing gp37-36, or the purified gp37-36 chimer, results in the assembly of linear multimers of increasing length.
  • the final rods are released by a brief incubation at high temperature (40-60° C, depending on the characteristics of the particular thermolabile P37 variant.)
  • the ratio of initiator to gp37-36 can be varied, and the size distribution of the rods is measured by any of the following methods: 1) Size exclusion chromatography; 2) Increase in the viscosity of the solution; and 3) Direct measurement by electron microscopy.
  • the P37-36 variants *P37-36 and P37-36* described in Example 3 cannot self- polymerize. This allows the staged assembly of rods of defined length, according to the following protocol: 1. Attach initiator P37 (preferably thermolabile) to a matrix. 2. Add excess *gp37-36 to attach and oligomerize as P37-36 homooligomers to the N-terminus of P37.
  • the linear dimensions of the protein rods in the batch will depend upon the lengths of the unit heterochimers and the number of cycles (n) of addition.
  • This method has the advantage of insuring absolute reproducibility of rod length and a homogenous, monodisperse size distribution from one preparation to another.
  • T4 37amA481 (Fisher, K. M.; Bernstein, H. MoI. Gen. Genet. 1970, 106, 139-150) was the mutant used to derive all phage strains discussed herein.
  • E. coli B40 (sul) (lab strain, courtesy of P. Strigini, Harvard Medical School, Boston) was used to grow and titer phage containing an amber mutation, and E. coli BB (suO) (McFaIl, E.; Stent, G. W. J. Gen. Microbiol. 1970, 18, 346-363) was used for all non-amber phages.
  • Primers cysF CATTAACGGACTTTTGAGA, Seq. ID No. 7
  • cysR TTCAATACGTCCAATAGTTT, Seq. ID No. 8
  • Adsorption rates were measured by using a single time point method (Adams, M. H. (1959) Bacteriophages (Interscience, New York)). Briefly, phage were incubated with log phase cells for a fixed time, usually 5 or 10 min at 37°C (within the phage eclipse period). At that time we diluted the phage/cell mixture into buffer saturated with chloroform to lyse the cells.
  • the pentaglycine coding segment in S ⁇ 1G5 was added to the cloned DNA in ⁇ 37S ⁇ l/T by using overlapping PCR primers (Sambrook, J. & Russel, D. W. (2001) Molecular Cloning (Cold Spring Harbor Lab. Press, Plainview, NY), 3rd Ed.).
  • Primers 37S ⁇ 1-1F GGCGATGGTGGCGGTGGCGGCAATGTACAATTTTACGCTG, Seq. ID No. 11
  • 37S ⁇ 1-1R TACATTGCCGCCACCGCCACCATCGCCATTTAATCTCAA, Seq. ID No. 12
  • flanking recF and 37B-2R primers were used with the flanking recF and 37B-2R primers to produce two modified half segments that were then recombined using the complementary ends to fuse the two segments and the flanking primers to amplify the whole segment.
  • the entire segment was then cloned into pGEM-T.
  • S ⁇ IUCS universal cloning site was creating by amplifying half segments of the S ⁇ 1 clone with primers 37S ⁇ 1-2F
  • Each primer contains a BsmBI and Kpnl site. The two half segments were joined using the Kpnl site to create a single segment with two BsmBI sites around the central Kpnl site inserted into the S ⁇ 1 junction.
  • This arrangement allows for the insertion (with an unambiguous orientation) of any double-stranded oligonucleotide with the correct cohesive ends.
  • any oligopeptide can be cloned into junction of the S ⁇ 1 deletion.
  • mAb from a 0.1 mg/ml stock
  • 500 ⁇ of diluted phage was added to 500 ⁇ of diluted phage and incubated the mixture for 30 min (unless otherwise indicated) at room temperature on a rotisserie mixer.
  • 4 ⁇ g of secondary antiserum from a 2 mg/ml stock
  • Fig. 3 A we used 1 ⁇ g of mAb, whereas 3 ⁇ g or the indicated amount was used for the remaining experiments.
  • Fig. 3D we mixed the peptide (EEYSAMRDQVMRTGE, Seq. ID No. 19) and mAb at a 10:1 molar ratio and incubated for 30 min at room temperature. The mAb/peptide mixture was then added to phage as described above.
  • Phage and phage/antibody complexes were stained with 1% phosphotungstate (pH 7) on carbon grids. Grids were examined at 100 kV by using a Philips CMlO transmission electron microscope. The final micrograph images were at a magnification of X 73,000.
  • the nanosensors of the instant invention contains specific PDLs to a paramagnetic nanoparticle and to a B. anthracis spore (the non-pathgenic Sterne strain).
  • B. anthracis display peptide binding sequences have already been identified. See Brigati, J. et al. "Diagnostic Probes for Bacills anthracis Spores Selected from a Landscape Phage Library" Clinical Chemistry 2004, 50, 1899-1906.
  • the problem with anthracis spore detection is that there is some background from the closely related B. cereus and B. thuringiensis strains. Our approach can ameliorate this problem by increasing avidity of the composite sensor by adding another one or more PDLs, spaced along each sensor, and thereby binding the strut parallel to the spore surface.

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Abstract

Des capteurs nanométriques utilisés dans la détection d'entités biologiques et chimiques formés à partir d'une partie du corps en forme de tige de la protéine (41), une particule magnétique (42) fixée sur la partie (41); et une fraction de liaison de substance à analyser (43) disposée sur la partie (41) à distance de la particule magnétique (42). La fraction de liaison de la substance à analyser se lie spécifiquement à la substance à analyser (44) afin de former un complexe de détection d'une substance à analyser. La partie du corps en forme de tige de la protéine peut être formée à partir d'une protéine à fibre caudale provenant d'un bactériophage homogène T ou de son dérivé. Comme la liaison de la substance à analyser au capteur va modifier la taille et la forme globales et, en conséquence, la capacité du capteur à se déplacer dans un échantillon liquide sous l'effet de l'application d'un champ magnétique, on peut considérer la liaison comme une modification de la susceptibilité magnétique ou de la durée de relaxation par rapport à celle d'un capteur en l'absence de la substance à analyser.
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EP1811040A1 (fr) * 2006-01-23 2007-07-25 Siemens Aktiengesellschaft Dispositif et procédé pour détecter un analyte
EP2428799A1 (fr) 2006-11-02 2012-03-14 ITI Scotland Limited Système de reconnaissance magnétique
US8697029B2 (en) 2002-04-18 2014-04-15 The Regents Of The University Of Michigan Modulated physical and chemical sensors
US8846331B2 (en) 2010-08-27 2014-09-30 The Regents Of The University Of Michigan Asynchronous magnetic bead rotation sensing systems and methods
US9068977B2 (en) 2007-03-09 2015-06-30 The Regents Of The University Of Michigan Non-linear rotation rates of remotely driven particles and uses thereof
US9797817B2 (en) 2012-05-03 2017-10-24 The Regents Of The University Of Michigan Multi-mode separation for target detection
US9816993B2 (en) 2011-04-11 2017-11-14 The Regents Of The University Of Michigan Magnetically induced microspinning for super-detection and super-characterization of biomarkers and live cells
US9983110B2 (en) 2013-11-04 2018-05-29 The Regents Of The University Of Michigan Asynchronous magnetic bead rotation (AMBR) microviscometer for analysis of analytes
WO2022087112A1 (fr) * 2020-10-21 2022-04-28 Athanor Biosciences, Inc. Détection à base de taille et quantification de bio-nanoparticules fonctionnelles

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Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8697029B2 (en) 2002-04-18 2014-04-15 The Regents Of The University Of Michigan Modulated physical and chemical sensors
EP1811040A1 (fr) * 2006-01-23 2007-07-25 Siemens Aktiengesellschaft Dispositif et procédé pour détecter un analyte
US8263331B2 (en) 2006-01-23 2012-09-11 Siemens Aktiengesellschaft Device and method for the detection of an analyte
EP2428799A1 (fr) 2006-11-02 2012-03-14 ITI Scotland Limited Système de reconnaissance magnétique
US9068977B2 (en) 2007-03-09 2015-06-30 The Regents Of The University Of Michigan Non-linear rotation rates of remotely driven particles and uses thereof
US8846331B2 (en) 2010-08-27 2014-09-30 The Regents Of The University Of Michigan Asynchronous magnetic bead rotation sensing systems and methods
US9816993B2 (en) 2011-04-11 2017-11-14 The Regents Of The University Of Michigan Magnetically induced microspinning for super-detection and super-characterization of biomarkers and live cells
US9797817B2 (en) 2012-05-03 2017-10-24 The Regents Of The University Of Michigan Multi-mode separation for target detection
US10724930B2 (en) 2012-05-03 2020-07-28 The Regents Of The University Of Michigan Multi-mode separation for target detection and cell growth monitoring
US12480846B2 (en) 2012-05-03 2025-11-25 The Regents Of The University Of Michigan Multi-mode separation for target detection and cell growth monitoring
US9983110B2 (en) 2013-11-04 2018-05-29 The Regents Of The University Of Michigan Asynchronous magnetic bead rotation (AMBR) microviscometer for analysis of analytes
WO2022087112A1 (fr) * 2020-10-21 2022-04-28 Athanor Biosciences, Inc. Détection à base de taille et quantification de bio-nanoparticules fonctionnelles

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