Innovative biotechnological systems for the detection of cellular or molecular analytes

Abstract

The present invention relates to a method for detecting cellular or molecular analytes, which uses two types of artificial phages, a "capture" phage (bait phage) and a "signal" phage (reporter phage), that are capable of selectively binding and thus recognizing the target analyte.

Description

[Technical field]

[0001] The present invention relates to a method for detecting cellular or molecular analytes, which uses two types of artificial phages, a "capture" phage (bait phage) and a "signal" phage (reporter phage), that are capable of selectively binding and thus recognizing the target analyte. [Background technology]

[0002] In the field of clinical diagnostic systems, molecular diagnostic systems, and other testing and diagnostic systems in general, there is always strong interest in developing innovative systems. Currently, the main analytical methods used to detect foreign analytes (viruses, molecular markers, toxins, proteins, nucleic acids, etc.) in cellular systems (microorganisms and eukaryotic cells) are mainly based on indirect detection systems, which, although highly sensitive (e.g. PCR, ELISA and other antibody tests), require qualified operators, waiting times for test results and centralized analysis.

[0003] Another aspect is the so-called POCT (Point of Care) techniques for developing innovative biotechnological and integrated biotechnological systems. In particular, such systems allow the performance of rapid analytical tests in non-clinical laboratories, which can be performed by non-qualified personnel or even by the patient himself. In particular, the growing research attention in the field of POCT is strongly driven by two different trends in modern society: (i) the general need to reduce high medical costs and (ii) the demand for better analytical solutions for early diagnosis and customized treatment. In addition to this, the demand for more accurate, sensitive, cheap and at the same time large-scale screening as a result of the pandemic explosion has become very important. These aspects are crucial to deal with efficient patient management, timely diagnosis, as well as rapid detection of infectious disease outbreaks, which will make it possible to deal efficiently with pandemics and epidemics.

[0004] Therefore, there is a continuing search for sensitive, selective and at the same time inexpensive direct readout analytical systems allowing the direct detection of molecular and / or cellular analytes, in particular microorganisms (e.g. bacteria and viruses), where said detection should be highly selective, specific and cost-effective. Summary of the Invention

[0005] The problem that the present invention aims to solve is therefore to provide a method for detecting analytes, such as, for example, cell lines (e.g. bacteria, parasites, eukaryotic cells), viruses, molecular markers (e.g. toxins, proteins, nucleic acids), preferably analytes of bioclinical interest, which is characterized by high sensitivity and specificity and can be used in a straightforward, rapid and cost-effective system.

[0006] This problem is solved by the method of the present invention as outlined in the appended claims, the definitions contained therein being an integral part of this disclosure. The applicant has now discovered a method for detecting cellular or molecular analytes using two types of artificial phages, namely, a bait phage and a reporter phage. Specifically, the phage contains at least one fusion peptide exposed on the major capsomer pVIII and capable of selectively binding to the target analyte. Then, the reporter phage, which exposes at least one recognition peptide on the minor capsomer pIII at the tip of the phage and exposes at least one marker for signal transduction on the major capsomer, binds to the analyte captured by the bait phage in the same manner. Such a detection system based on two types of artificial phages can be defined as a "molecular sandwich".

[0007] Thus, in a first aspect, the present invention provides a method for producing a method for treating a cancer cell comprising the steps of: a) contacting a sample containing an analyte with a first phage M13 (bait phage) bound to a suitable support and comprising at least one peptide or polypeptide or fusion protein specific for recognizing the analyte, said peptide or polypeptide or fusion protein being exposed on the protein pVIII, thereby achieving the formation of a first complex ("bait phage-analyte" complex) comprising the first phage M13 bound to the analyte; b) separating the first phage M13 from the portion of the sample that did not bind to the first phage M13 in the previous step a); c) adding to the first phage M13 a second phage M13 (reporter phage) comprising at least one peptide or polypeptide or protein for specific recognition of the analyte fused on the protein pIII and at least one marker conjugated to the capsid of the second phage M13, said marker being selected from a fluorophore, a chromophore, an electrochemically active species or an electrochemiluminescence active species, thereby achieving the formation of a second complex ("bait phage-analyte-reporter phage" complex) comprising the first phage M13 and the second phage M13 bound in a sandwich manner to the analyte; d) performing a wash to remove the second phage M13 that did not bind to the analyte; e) determining the induction signal generated by the marker of the second phage M13 of the second complex; The present invention relates to a method comprising the steps of:

[0008] Advantageously, the method according to the invention allows selective, sensitive and direct detection of target analytes. In particular, said method can be used for the direct detection of infectious microorganisms (e.g. bacteria, parasites and viruses, e.g. SARS-CoV-2). Other advantageous examples include the direct detection of human eukaryotic cells, as in the case of mutations due to pathologies, such as oncological pathologies, and of analytes of different molecular nature (e.g. proteins, molecular markers, toxins, nucleic acids, etc.). Advantageously, the method according to the invention offers the possibility of multiplexed detection for the integrated analysis of several analytes simultaneously. Also, another advantageous aspect is that this type of detection is not expensive. Finally, the implementation of the method according to the invention for POCT is also possible.

[0009] Further features and advantages of the method of the invention will become apparent from the description of exemplary embodiments of the invention, which are provided as indicative examples of the invention itself. [Brief description of the drawings]

[0010] [Figure 1]FIG. 2 is a schematic diagram of the mechanism of action of the method according to the invention. [Diagram 2] An image of the molecular sandwich "bait phage + analyte + reporter phage" is shown. [Diagram 3] Further images of the molecular sandwich "bait phage + analyte + reporter phage" are shown. [Figure 4] Further images of the molecular sandwich "bait phage + analyte + reporter phage" are shown. [Diagram 5] Further images of the molecular sandwich "bait phage + analyte + reporter phage" are shown. [Figure 6] Further images of the molecular sandwich "bait phage + analyte + reporter phage" are shown. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] For purposes of the present invention, definitions of certain terms used in the specification and appended claims are provided below. The term "cellular or molecular analyte" refers to systems such as, for example, cell systems (microorganisms, parasites, eukaryotic cells, etc.), viruses, molecular markers (toxins, proteins, nucleic acids, etc.), preferably of bioclinical interest.

[0012] The object of the present invention is a method for detecting an analyte in a sample, said analyte comprising: a cell line selected from a microorganism, a parasite, a eukaryotic cell, a bacterium, a virus, or A molecular marker selected from a toxin or a protein; is selected from The method comprises: a) contacting the sample with a first phage M13 (bait phage) bound to a suitable support and comprising at least one peptide or polypeptide or fusion protein specific for recognizing the analyte, said peptide or polypeptide or fusion protein being exposed on the protein pVIII, thereby achieving the formation of a first complex ("bait phage-analyte" complex) comprising the first phage M13 bound to the analyte; b) separating the first phage M13 from a portion of the sample that did not bind to the first phage M13; c) adding to the first phage M13 a second phage M13 (reporter phage) comprising at least one peptide or polypeptide or protein for specific recognition of the analyte fused on the protein pIII and at least one marker conjugated to the capsid of the second phage M13, said marker being selected from a fluorophore, a chromophore, an electrochemically active species or an electrochemiluminescence active species, thereby achieving the formation of a second complex ("bait phage-analyte-reporter phage" complex) comprising the first phage M13 and the second phage M13 bound in a sandwich manner to the analyte; d) performing a wash to remove the second phage M13 that did not bind to the analyte; e) determining the induction signal generated by the marker of the second phage M13 of the second complex; The method includes:

[0013] The above steps are performed in the order given above. Preferably, the first phage M13 (bait phage) is an artificial form of M13 exposing at least one fusion peptide, preferably a fusion with the phage protein "major coat protein pVIII" capable of selectively binding to the analyte. Preferably, the at least one fusion peptide is exposed on the capsid of the first phage M13 in a copy number of the order of tens or hundreds, up to several thousand.

[0014] The second phage M13 (reporter phage) is the phage M143 engineered in a manner known to those skilled in the art to expose at least one peptide or polypeptide or fusion protein capable of selectively binding to the analyte, preferably a fusion with the "minor coat protein pIII" of the phage. The protein "minor coat protein pIII" is a protein of the capsid of said second phage M13.

[0015] In relation to the marker, the induced signal generated by the marker can be determined. Such a signal determination can be carried out by optical or electronic detection of the analyte. In particular, at least one marker can be detected by photometry, fluorometry, photoluminescence, thermoluminescence, chemiluminescence, electrochemiluminescence, voltammetry and other electrochemical techniques. Indeed, after passing step c), the resulting "capture" signal of the analyte can be detected by optical or electronic sensors, depending on the marker used. According to a preferred embodiment, the marker conjugated to the capsid of the second phage M13 is a fluorophore, a chromophore, an electrochemically active species or an electrochemiluminescence active species. According to a preferred embodiment of the present invention, the marker is a fluorophore, the fluorophore being preferably CF594 (Biotium).

[0016] According to a preferred embodiment of the invention, the analyte is a bacterium, preferably a Gram-negative bacterium, more preferably P. aeruginosa. Preferably, at least one peptide or polypeptide or fusion protein of step a) has the sequence of SEQ ID NO: 1 (QRKLAAKLT). Preferably, at least one peptide or polypeptide or modality recognition protein of step c) has the sequence of SEQ ID NO: 2 (KLAKLAKKLAKLAK).

[0017] According to a preferred embodiment of the present invention, when the analyte is Pseudomonas aeruginosa, the at least one peptide or polypeptide or fusion protein of step a) has the sequence (QRKLAAKLT) of SEQ ID NO: 1. Preferably, said at least one fusion peptide is exposed on the capsid of the first phage M13 in a copy number of the order of tens or hundreds, up to several thousand.

[0018] According to a preferred embodiment of the invention, when the analyte is Pseudomonas aeruginosa, the at least one peptide or polypeptide or phage recognition protein of step c) has the sequence of SEQ ID NO: 2 (KLAKLAKKLAKLAK).

[0019] According to a preferred embodiment of the invention, when the analyte is Pseudomonas aeruginosa, at least one peptide or polypeptide or fusion protein in step a) has the sequence of SEQ ID NO:1 (QRKLAAKLT) and at least one peptide or polypeptide or phage recognition protein in step c) has the sequence of SEQ ID NO:2 (KLAKLAKKLAKLAK).

[0020] According to a preferred embodiment, a linker can be used. Advantageously, the linker confers mobility characteristics to the peptide. Preferably, the linker is GGGS. In a preferred embodiment of the method according to the invention, a support is used, for example a magnetic microsphere (MNP), which is functionalized with a bait phage (thus obtaining an MNP-bait phage conjugate), said phage containing at least one fusion peptide exposed on the major capsomer pVIII and capable of selectively binding the target analyte. An exemplary but non-limiting procedure of functionalization is also given below. Thus, in the detection method according to the invention, a support functionalized with a first phage M13 can be used. Preferably, the support is a magnetic microsphere (MNP). Here, the term "magnetic microsphere" refers to a particle, generally referred to in the art as a magnetic microparticle MNP, capable of immobilizing one or more phages. As will be understood by those skilled in the art, said particles can also be referred to herein using the term sphere / spheres and corresponding terms (e.g. microspheres) interchangeably. Such particles can preferably be inorganic particles, such as ferrite, and can be functionalized with one or more phages. As a result of said functionalization, a biosensor can be obtained. In particular, microspheres have the advantage of being three-dimensional structures with a higher surface area to volume ratio than planar surfaces, providing more binding sites for targets. Other advantages are that they are easy to handle in fluids and are cost-effective. According to a preferred embodiment, selected phages exposing active recognition peptides fused to protein pVIII are covalently attached onto magnetic microspheres. This is advantageous for use in direct capture, washing and rapid concentration of cells, viruses or proteins in test samples.

[0021] According to a preferred embodiment, when the support used for the first phage M13 consists of magnetic microspheres, the step of separating the first phage M13 from the sample portion not bound to the first phage M13 is carried out by magnetic capture of the magnetic microspheres.

[0022] According to a further preferred embodiment, the support for the first phage M13 is an electrode, a polymer or a silicon-based surface, in which case the step of separating the first phage M13 from the sample portion not bound to the first phage M13 is carried out by washing.

[0023] A preferred application of the method of the invention aims at the direct detection of microbial cells, with particular reference to infectious microorganisms (bacteria and viruses), such as pathogenic Gram-negative bacteria (e.g. species on the WHO "priority list") or viruses like SARS-CoV-2. Such a method can also be extended to liquid biopsies with the possibility of capturing, enriching and directly detecting eukaryotic cells, especially cancer cells. In this case, the selectivity of the method concerns the specificity of the artificial bait and reporter phages for certain tumor markers exposed on the cell surface. Such a method can also be extended to analytes of molecular nature, such as toxins, proteins or nucleic acids, especially when these are considered biomarkers of certain pathologies. The development of such "phage probes" and the flexibility of the analytical platform presented accordingly are favored by the great adaptability of the phage display scheme, allowing the selection of peptide and antibody libraries (scFvs, single domain antibodies, etc.) by biopanning after immobilization of the protein of interest (either wild type or recombinant) on the aforementioned supports.

[0024] It should be understood that the above is illustrative and not limiting, and that those skilled in the art will recognize that modifications can be made without departing from the scope of the invention. EXAMPLES

[0025] Reagents used and their preparation

[0026] [Table 1]

[0027] Functionalization experimental procedure 50 μl of tosyl-activated Dynabeads M-280 (Invitrogen Cat. No. 142.03) were placed in a round-bottom Eppendorf tube and washed twice with 500 μl of borate buffer (0.1 M borate buffer pH 9.5) for 5 min with gentle agitation on a wheel. After 10 min of magnetic collection of beads, the buffer was discarded and the beads were resuspended in 50 μl of borate buffer. 12 30 μl of bait phage from TU / ml stock, i.e. 3x10 6 Phage clones were added, followed by 60 μl of borate buffer and 60 μl of ammonium sulfate buffer (3M pH 7.4). The tubes were incubated at 37°C for 24 hours with gentle agitation on a rotating tumbler mixer at 30° inclination. The beads were separated on a magnetic device for 10 minutes, the supernatant was discarded, and the beads were washed twice with 500 μl of PBS buffer pH 7.4 + 1% BSA for 5 minutes with agitation on a wheel. Then, 500 μl of blocking buffer (PBS pH 7.4 + 4% BSA) was added, and the beads were agitated on a wheel for 2 hours at room temperature (RT). The beads were separated on a magnetic device for 10 minutes, the supernatant was discarded, and the beads were washed twice with 500 μl of PBS buffer pH 7.4 + 1% BSA for 5 minutes on a rotating tumbler mixer. Finally, the beads were resuspended in 200 μl of PBS and left at 4°C (Dynabeads-bait phage). Three different stocks were prepared.

[0028] ELISA test to confirm the functionalization of Dynabeads with bait phages To confirm proper functionalization of the beads with the bait phages, an ELISA test using anti-phage M13 antibody was performed. Briefly, 20 μl of functionalized beads were washed twice with 500 μl of washing buffer (PBS pH 7.4 + 0.5% Tween 20) for 5 min on a wheel. The beads were resuspended in 100 μl of PBS, 70 μl of anti-M13 HPR antibody (1:2500 in PBS + 0.1% BSA + 0.5% Tween 20) was added, and the beads were then incubated for 1 h at 37 °C on a rotating tumbler mixer. Afterwards, 5 washes were performed with 500 μl of PBS + 0.5% Tween 20. In the last step, the beads were resuspended in 250 μl of TMB and incubated for 20 min on a wheel in the dark at room temperature (RT). After complete color development, the reaction was quenched with 31 μl of 6N H2SO4. The beads were placed on a magnet for 10 minutes, after which the entire solution and 200 μl of the 1:10 dilution were read at 450 nm.

[0029] Results obtained:

[0030] [Table 2]

[0031] Capture assay of Pseudomonas aeruginosa using the phage system Dynabeads / phage-pVIII Capture Test For the capture and detection tests, P. aeruginosa ATCC 27853 isolated on cetrimide agar medium was grown in 5 ml of LB. The bacteria were collected by centrifugation at 8000xg for 10 min and resuspended in an equal volume of PBS. The bacteria were diluted with PBS and the cultured bacteria stock (10 8 20 μl of Dynabeads-bait phage were washed twice with 500 μl of PBS for 5 min on a wheel, then placed on a magnet for 10 min, the supernatant discarded, resuspended in 1 ml of PBS, and mixed with 100 μl of the bacterial suspension for 10 min. 8 cells / ml (final concentration 10 7 cells) or 100 μl of PBS (negative control) was added.

[0032] The samples were placed on a tilt wheel at 37° C. for 30 minutes. After separation of the beads-bait phage containing any captured bacteria on a magnetic device for 10 min, the supernatant was removed and the beads-phage-bacteria complexes were resuspended in 50 μl of PBS and used for recognition by phage-pIII-CF594. The uncaptured bacteria present in the supernatant were titrated by plate dilution and the CFU counts were counted on cetrimide agar medium to evaluate the bacterial capture efficiency of Dynabeads functionalized with bait phages. All tests were performed in triplicate. The capture efficiency of P. aeruginosa cells by Dynabeads-bait phage in the tests performed (calculated as the ratio of input bacteria number - number of bacteria in the supernatant = number of captured bacteria) was 90% on average.

[0033] Engineering of a reporter phage (phage-pIII-CF594) Construction of Gram-negative bacterial reporter phages A sequence encoding the peptide KLAKLAKKLAKLAK, capable of binding to the outer membrane of Gram-(-) bacteria, was cloned in frame into the short linker GGGS upstream of gene p3 of the phagemid pSEX81 (ProGen) for pentavalent display at the tip of the detector phage pIII. Exponential phase cultures of E. coli TG1 transformed with the recombinant phagemid were then superinfected with the helper phage Hyperphage (ProGen) to allow the production of phage-pIII virions redirected for recognition in Gram-(-) bacteria. After purification by precipitation with a NaCl / PEG solution, phage-pIII was diluted with PBS pH 7.4 for approximately 10 min and then diluted with PBS pH 7.4 for approximately 10 min. 13 The redirected phage-pIII was conjugated with the fluorophore CF594 and resuspended at a target concentration of phage / ml.

[0034] Conjugate of reporter phage with fluorescent dye molecule CF594 The method for binding the imaging agent, CF594, to the capsid of the detector phage pIII is as follows: A stock solution of 10 mM CF594 succinimide ester in DMSO is prepared at room temperature.

[0035] Add 10 μl of the stock solution to 1 ml of reporter phage-pIII in 0.1 M bicarbonate buffer, pH 8.3, under constant magnetic stirring. The succinimidyl ester group of the dye reacts with the amine group of the phage capsid protein to form a stable amide bond. Leave the mixture stirring overnight at room temperature.

[0036] The functionalized phage are separated from the non-conjugated fraction of CF594 by dialysis in PBS buffer (cut-off 14 kDa). Three cycles of dialysis for 8 h each were performed using 1 ml of the solution of the bioconjugate to be dialyzed and 500 ml of PBS.

[0037] The phage was quantified by reading the UV-Vis absorbance spectrum of the phage, determining the value at wavelengths from 269 nm to 320 nm, and then applying the following formula. Phage concentration (virions / mL) = (A269-A320)*6*10^16 / bp phage genome = 2.04E+13 To quantify phage-conjugated CF594, the following formula was applied:

[0038] Concentration of CF594 (microM) = A594 / epsilon594 = 8.91 Given the concentration of CF594 and the concentration of phage (virions / ml), the ratio of CF594 molecules / phage is obtained, which is equal to 264.

[0039] Validation of reporter phage pIII-CF594 redirection by cytofluorometry The redirection of phage-pIII-CF594 into Pseudomonas aeruginosa (and other Gram-negative bacteria) was verified by performing a cytofluorometric assay using a BioRad S3e Cell Sorter.

[0040] Phage-pIII-CF594 10 10 10 virions of Pseudomonas aeruginosa PA14 resuspended in 1 ml of PBS pH 7.4 9 The cells were incubated with PA14 for 20–30 min. Non-redirected phage M13K07 (New England Biolabs) conjugated to CF594 was used as a negative control for cytofluorometric analysis. The MFI (mean fluorescence intensity) detected with phage pIII-CF594 was increased more than four-fold over the control M13K07-CF594, proving that the detection phage was redirected to PA14. Cytofluorometric analysis also confirmed that one-third of the P. aeruginosa population showed an approximately one order of magnitude (1 Log) increase in fluorescence intensity after the addition of reporter phage-pIII-CF594. This is an indication of the ability of the detection phage to mediate the accumulation of multiple fluorophores in the vicinity of Gram(-) bacterial cells.

[0041] Capture and detection test using double phage system (Dynabeads / bait phage-pVIII + reporter phage-pIII-CF594) Labeling with reporter phage pIII-CF594 To the bead-phage-bacteria complexes resuspended in 50 μl PBS and to the negative control samples (no bacteria), 2 μl of phage-pIII-CF594 (labeled stock 1:1, 2.04E+13 phage / ml, 0.91 μM CF594, 246 fluorophores / phage) was added and placed on a rotating mixer for 40 min at 37°C, and 1 μl of DAPI (stock 10 mg / ml) or SYTO9 was added during the last 5 min of the reaction to label the captured bacterial cells. Each sample was placed on a magnet for 10 min, the supernatant was discarded, and a gentle wash was performed with 100 μl of PBS at room temperature for 5 min without agitation. Afterwards, the sample was placed again on a magnet for 10 min, the supernatant was discarded, and a gentle resuspension was performed with 50 μl of PBS. Ten microliters of each sample was placed on a slide, the samples were viewed under an Olympus fluorescence microscope (filter 540 / 605 lens 40x and 100x), and images were captured with a CCD camera.

[0042] Comments on the results The results obtained by labeling of captured bacteria with reporter phage-pIII-CF594 show specific recognition of phage pIII on P. aeruginosa complexed on Dynabeads functionalized with the bait phage.

Claims

1. 1. A method for detecting an analyte in a sample, the method comprising: a cell line selected from a microorganism, a parasite, a eukaryotic cell, a bacterium, or a virus; or a molecular marker selected from a toxin, a protein, or a nucleic acid; is selected from The method comprises: a) contacting the sample with a first phage M13 (bait phage) bound to a suitable support and comprising at least one peptide or polypeptide or fusion protein specific for recognizing said analyte, said peptide or polypeptide or fusion protein being exposed on the protein pVIII, thereby achieving the formation of a first complex ("bait phage-analyte" complex) comprising the first phage M13 bound to said analyte; b) separating the first phage M13 from the portion of the sample that did not bind to the first phage M13; c) adding to said first phage M13 a second phage M13 (reporter phage) comprising at least one peptide or polypeptide or protein for specific recognition of said analyte fused on protein pIII and at least one marker conjugated to the capsid of said second phage M13, said marker being selected from a fluorophore, a chromophore, an electrochemically active species or an electrochemiluminescently active species, thereby achieving the formation of a second complex comprising the first phage M13 and the second phage M13 sandwich-bound to said analyte; d) performing a wash to remove the second phage M13 that did not bind to the analyte; e) determining the induction signal generated by the marker of the second phage M13 of the second complex; A method comprising:

2. The method of claim 1 , wherein the analyte is a bacterium.

3. The method of claim 2 , wherein the analyte is a gram-negative bacterium.

4. 4. The method of claim 3, wherein the analyte is the gram-negative bacterium Pseudomonas aeruginosa (P. aeruginosa).

5. 5. The method of claim 4, wherein at least one peptide or polypeptide or fusion protein of step a) has the sequence of SEQ ID NO: 1 (QRKLAAKLT).

6. 5. The method of claim 4, wherein the at least one peptide or polypeptide or phage recognition protein of step c) has the sequence of SEQ ID NO: 2 (KLAKLAKKLAKLAK).

7. The method of claim 1 , wherein the marker is a fluorescent dye molecule.

8. 8. The method of claim 7, wherein the fluorophore is CF594 (Biotium).

9. The method according to any one of claims 1 to 8, wherein the support comprises magnetic microspheres (MNPs), and the magnetic microspheres are functionalized with the first phage M13.

10. 10. The method of claim 9, wherein the step of separating the first phage M13 from the portion of the sample that did not bind to the first phage M13 is performed by magnetic capture of magnetic microspheres.

11. The method of claim 1 , wherein the support is an electrode, a polymer, or a silicon-based surface.

12. 12. The method of claim 11, wherein the step of separating the first phage M13 from the portion of the sample that did not bind to the first phage M13 is performed by washing.

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