WO2010142532A1

WO2010142532A1 - Modified bacteriophage, biosensor containing same, and method of use

Info

Publication number: WO2010142532A1
Application number: PCT/EP2010/057279
Authority: WO
Inventors: Joris Proost; Ronny Santoro; Patrice Soumillion; Denis Flandre; Geneviève DESCHUYTENEER
Original assignee: Universite Catholique de Louvain UCL
Current assignee: Universite Catholique de Louvain UCL
Priority date: 2009-06-12
Filing date: 2010-05-26
Publication date: 2010-12-16
Anticipated expiration: 2011-12-12

Abstract

The invention relates to bacteriophages comprising a modified coat protein capable of binding to a metal oxide surface. The invention also relates to methods for detecting and/or quantifying targets in a sample using same.

Description

Modified bacteriophage, biosensor containing same, and method of use

[FIELD OF THE INVENTION]

This invention is directed to the field of biosensors and more particularly to bacteriophage having a modified coat protein.

[BACKGROUND OF THE INVENTION]

Surface design for biologically oriented micro- and nano-electronic structures is currently shifting from chemically modified synthetic surfaces towards interfacial layers of genetically engineered biomaterials, like polypeptides, nucleic acids or other macromolecules [I]. This new approach is motivated by the fact that traditional molecular linkers, like thiol- or silane-based self-assembled monolayers, are relatively non-specific in their reactivity with the inorganic surface, and generally do not convey specific biorecognition properties to the synthetic micro- and nanoscale devices [2]. On the contrary, genetically engineered polypeptides have recently been shown to exhibit specific affinity to several Si-based inorganic surfaces [3,4], on top of their specific molecular recognition and genetic activity [5]. An essential aspect of biosensor development is the search for specific molecular linkers (mediators) for the attachment and/or recognition of the targeted biological species in solution. While thiol- and silane-based self-assembled monolayers are generally being used as mediators, the use of bacteria and phages has recently been reported as well, since they can be genetically engineered to exhibit specific affinity with several metallic or Si-based inorganic surfaces. For integrated biosensing devices, it was recently shown that metallic oxides may represent significant advantages over metallic or silicon oxides thin films, since it provides for a more reliable passivating encapsulation of the device in some of the corroding biological media encountered for diagnostic applications [6]. [SUMMARY OF THE INVENTION]

The inventors have now found that specific foreign peptide sequences may modify a filamentous phage, when inserted in a coat protein of said phage, in order to convey to the latter a specific affinity towards metallic oxide surfaces. The invention thus relates to bacteriophages comprising a coat protein modified with at least one amino acid sequence, wherein said amino acid sequence comprises 4 to 17 amino acids, and wherein the modified coat protein is capable of binding to a metal oxide surface. In one embodiment, said amino acid sequence comprises 5 to 12 amino acids, more preferably 8 amino acids.

Typically, said amino acid sequence is inserted in place of native amino acids of a coat protein of the bacteriophage. Advantageously, the coat protein is the pVIII coat protein. According to a preferred embodiment, the bacteriophage is a filamentous bacteriophage mutant, preferably a M13, fd or fl mutant. The native sequence of the pVIII coat protein of M13 mutant is the following: AEGDDPAKAAFDSLQASATEYIGYAWAMVVVIVGATIGIKLFKKFTSKAS (SEQ ID NO: 19).

In an embodiment of the invention, the three native amino acid EGD (in positions 1, 2 and 3 in SEQ ID NO: 19) are replaced by said amino acid sequence of 4 to 17 amino acids, preferably 5 to 12 amino acids, more preferably 8 amino acids, according to the invention. Consequently, the phage displays a pVIII protein wherein the three native amino acid EGD are replaced by an amino acid sequence of 4 to 17 amino acids, preferably 5 to 12 amino acids, more preferably 8 amino acids. According to an embodiment of the invention, at least one amino acid sequence is selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO:11, SEQ ID NO: 12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18. Accordingly, the invention also relates to bacteriophages comprising a modified pVIII coat protein, wherein said modified pVIII coat protein has an amino acid sequence selected from the group consisting of SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36 and SEQ ID NO:37. Typically, these bacteriophages are filamentous bacteriophages. Particularly, these bacteriophages are selected from Ml 3, fd and fl mutants.

The bacteriophage according to the invention may further comprise genetically modified pill and/or pIX coat proteins, capable to selectively recognize targets, such as for example pathogens, small molecules, proteins, viruses, ions, microorganisms or the like. Typically, the bacteriophages according to the invention are genetically engineered.

In one embodiment, the bacteriophages according to the invention are biotinylated. The invention also relates to nucleic acid sequences encoding an amino acid sequence selected from the group consisting of SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36 and SEQ ID NO:37. It is another object of the invention to provide a biosensor comprising a bacteriophage according to the invention. According to an embodiment, the biosensor further comprises a substrate at least partially coated with at least one metal oxide. Preferably, the metal oxide is AI2O3, Ta₂Os or TiO₂. This invention also relates to a method of detecting and/or quantifying targets in a sample, comprising the following steps: - contacting a sample with a bacteriophage according to the invention, said bacteriophage being or not being immobilized on a metal oxide surface,

- in the event that it was not immobilized on a metal oxide surface, further immobilizing the bacteriophage on a metal oxide surface;

- then detecting and/or quantifying the complex formed by interaction of said target and said bacteriophage.

[DETAILED DESCRIPTION OF THE INVENTION] As described above, the invention provides bacteriophages wherein a coat protein of said bacteriophage is modified with at least one amino acid sequence, said amino acid sequence comprising 4 to 17 amino acids, the modified coat protein being capable of binding to a metal oxide surface. Without being linked to any theory, the Applicant thinks that the presence of negatively charged amino acid, i.e. either alanine (A), asparagine (D) or glutamine (E), next to the ends of the inserted amino acid sequence may have some influence in the selective and/or specific binding to the metal oxide surface. Preferably, the amino acid sequence is inserted between a residue (A) and a residue (D) or (E), of the native coat protein.

It is furthermore preferred that the amino acid sequence has a positively charged, basic amino acid, i.e. either lysine (K) or arginine (R), at about the middle of the sequence. Without wanting to be bound to any theory, it is believed that the presence of a positively charged, basic amino acid at about the middle of the amino acid sequence causes the peptide to kink at about the middle due to electrostatic repulsion and thus enhances the direction of the terminal acidic, negatively charged amino acid or acids to the metal oxide surface. According to one embodiment, the amino acid sequence comprises from 4 to 17, preferably from 5 to 12, preferably from 6 to 10, more preferably from 7 to 9, and even more preferably about 8 amino acids. Very good results were obtained when using an amino acid sequence with 8 amino acids. The most preferred peptides present a positively charged, basic amino acid, such as for example lysine (K) or arginine (R) in the middle of the sequence, typically on the 4^th or 5^th position when the amino acid sequence is of 8 amino acids. As set forth above, it is believed that the presence of a positively charged, basic amino acid in middle positions may cause the peptide to kink in the middle due to electrostatic repulsion and thus enhances the direction to the metal oxide surface of the acidic, negatively charged amino acids, bordering to the sequence, i.e. present just before and just after the 4-17 amino acid sequence. Moreover, the presence and number of polar, non-charged groups (especially S, T, Q, N) and non-polar ones (especially A, V, P) may be used to further refine adsorption behavior.

Suitable metallic oxides include, but are not limited to, binary or ternary oxides of Al, Ta, Ti, Zn, Sn, Nb, Zr, Sr, W, Ba. Preferably, for integrated biosensor applications, the metal oxide is selected from the group consisting of the valve metals, such as for example Al, Ta, Ti, Zr, W, more preferably AI₂O3, Ta₂Os, and/or TiO₂. The metal oxide may be amorphous or under crystalline form.

In a biological medium these metal oxide surfaces may become protonated, presenting a positive surface charge density in the biological medium. For example, for AI₂O₃, according to published values for the point of zero charge (PZC) of Al₂O₃ (PZC ≡ 9) [12], it is to be expected that at a neutral pH, the alumina surface becomes protonated. As a result, the specific affinity of the amino acid sequences with metal oxide surfaces, according to the invention, seems to be governed by both structural features and electrostatic interactions. Suitable bacteriophages include, but are not limited to, filamentous bacteriophages, for example of the inoviridae family, such as for example M13 or fd or fl mutants. The bacteriophage mutant may also comprise a further genetic modification on the same or another coat protein so as to render it capable of selectively recognizing in-situ specific targets in solution or suspension. Preferably, a further genetic modification occurs on the pill or pIX coat protein. According to an embodiment of the invention, the bacteriophages may be immobilized on a metal oxide surface, due to the affinity of the modified PVIII coat protein thereto. This immobilization may either be an ex-situ immobilization, i.e. before contact of the bacteriophage with a target biological solution, or an in- situ immobilization of the bacteriophage contained in a biological medium by using the diagnostic event itself, i.e. capturing of the target by the phage, followed by phage immobilization on the metal oxide surface. The invention also relates to nucleic acid sequence encoding an amino acid sequence selected from the group consisting of SEQ ID NO:20, SEQ ID N0:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID N0:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36 and SEQ ID NO:37.

The term "nucleic acid sequence" as used in this document includes genomic DNA, cDNA, synthetic DNA, and RNA. Preferably it means DNA, more preferably cDNA sequence coding for a fragment according to the invention. The nucleotide sequence may be of genomic or synthetic or recombinant origin, which may be double-stranded or single-stranded whether representing the sense or anti- sense strand.

The invention also relates to bacteriophages comprising a nucleic acid according to the invention.

The invention also relates to biosensors comprising a bacteriophage according to the invention. According to an embodiment, the biosensor further comprises a substrate at least partially coated with a metal oxide. Preferably, the metal oxide is AI₂O3, Ta₂Os or TiO₂. Preferably, the substrate is at least partially coated with a thin dense AI₂O3, Ta₂Os or TiO₂ film, having preferably a thickness of 50-1000 nm, preferably about 100 nm.

According to an embodiment, the biosensor includes a bacteriophage according to the invention, preferably M13, having a coat protein, preferably pVIII, modified with at least one amino acid sequence selected among SEQ ID No.l to SEQ ID No.16 and a substrate at least partially coated with AI₂O3. Typically, the biosensor includes a bacteriophage comprising a pVIII coat protein having an amino acid sequence selected from the group consisting of SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34 and SEQ ID NO:35. According to another embodiment, the biosensor includes a bacteriophage according to the invention, preferably Ml 3, having a coat protein, preferably p VIII, modified with at least one amino acid sequence selected among SEQ ID No.17 to SEQ ID No.18 and a substrate at least partially coated with Ta₂Os. Typically, the biosensor includes a bacteriophage comprising a pVIII coat protein of SED ID NO:36 or SEQ ID NO:37.

In a preferred embodiment, the bacteriophage mutant is a M 13 mutant. Preferably, it also comprises a genetic modification on the pill and/or pIX coat protein of Ml 3, so that it can be used to selectively detect in-situ specific molecules in solution. The biosensor according to the invention may further comprise a substrate said substrate comprising a metal oxide surface.

The substrate may be any suitable conventional substrate which comprises a metal oxide surface. One example of a suitable substrate is a silicon wafer. The metal oxide surface may cover the surface of the substrate totally or partially. In a preferred embodiment the metal oxide surface covers the surface of the substrate only partially. It may for example be applied to predetermined areas of the substrate only. As the biosensor of the invention selectively and specifically to the metal oxide surface, parasitic effects during transducing and read-out are greatly reduced. The metal oxide surface may be obtained by any suitable method known in the art, such as anodic oxidization.

According to an embodiment, the invention also relates to an array including a bacteriophage according to the invention.

According to an embodiment, the invention also relates to a kit including a bacteriophage according to the invention. This invention also relates to a method of detecting and/or quantifying targets in a sample, comprising the following steps:

- contacting a sample with a bacteriophage according to the invention, said bacteriophage preferably being mutants of the inoviridae family, such as M 13 or fd or fl mutants, and preferably comprising a further genetic modification on the same or another coat protein, preferably on the pill or pIX coat protein, so as to render it capable of selectively recognizing specific targets, such as pathogens, microorganisms or small proteins or the like, in solution or suspension

- simultaneously or sequentially, contacting the bacteriophage to a metal oxide surface in order to have the bacteriophage interacted with the metal oxide surface

- then detecting the complex formed by interaction of said target and said bacteriophage.

According to an embodiment, the phage may be first bound (or immobilized) on the metal oxide surface of the substrate and then contacted with the target- containing solution or suspension.

Alternatively, the phage is first contacted with the target-containing solution or suspension and then bound on the metal oxide surface of the substrate.

As used herein, the term

"sample" refers to any sample containing material, such as food matrix, biological fluid (serum, urine...), biopsy material, environmental sample such as water as sewage water, freshwater, marine coastal water, ground water for example; - "microorganisms" refers to any viable organism of microscopic size, including bacteria, cyanobacteria, chlamydiae, fungi, algae, protozoa and viruses;

"pathogen" refers to any infectious agent, or germ, susceptible to cause disease or illness to a living being, including human; small molecules means small proteins of at most 100 amino acids, analytes having a molecular weight below 100 kDa; "target" refers to microorganisms, pathogens, small molecules, viruses, ions, proteins or the like;

"binding", "to bind" (or any form of the verb to bind) refers to any detectable selective and/or specific affinity, and may apply to the specific affinity of the amino acid sequences to metal oxide surfaces, and/or to any recognition of a target by the phage. The affinity of the amino acid sequences to the metal oxide surface may result in immobilization of the bacteriophage according to the invention;

"genetically engineered" and "genetically modified" are used herein interchangeably;

- A bacteriophage comprising "a coat protein" refers either to a bacteriophage comprising a single copy of said coat protein or comprising several copies (2, 5, 10, 100, 1000, 10 000, etc.) copies of said coat protein.

The present invention will be better understood with reference to the following examples and figure 1. These examples are intended to representative of specific embodiments of the invention, and are not intended as limiting the scope of the invention.

[FIGURE]

Fig. 1: ELISA analysis of the affinity of a bacteriophage having pVIII modified with SEQ ID NO:3, previously purified and biotinylated, for an AI2O3 surface.

[EXAMPLES]

EXAMPLE 1: Phage binding to an anodic Al?Ch surface using phage display technique [7]

Anodic AI₂O₃ surfaces were obtained by anodizing 400nm thick pure aluminium thin films, evaporated onto oxidised 3" silicon wafers. Thin film anodizing was carried out galvanostatically in 0.01 M ammonium octahydrate pentaborate at 6 mA/cm², according to the procedures described by Vanovermeere et al. [8], leading to a 100 nm thick dense AI₂O3 film.

As to the selection of amino acid sequences, a combinatorial library of random peptides was used, containing 8 amino acids inserted in the pVIII coat protein of M 13 bacteriophages in place of native EGD.

Before starting the bio-panning protocol, which has already been described in detail by Soumillion [9], alumina surfaces were washed three times with 1 ml of Tris-HCl buffered saline (TBS). A round of selection consisted of incubating an equal amount of phages (1.5 1012) in a TBST buffer (0.14 M NaCl, 0.1M Tris-HCl, 0.1% v/v of Tween 20, pH = 7.25) for 1 hour. The non-bound phages were then collected, and the alumina surfaces were washed 10 times with TBST. An elution solution (0.2 M glycine-HCl/ lg/1 BSA, pH 2.2) was then added and incubated for 8 min in order to recover bound phages, followed by neutralisation in IM Tris-HCl at pH 9. This elution procedure was repeated twice. Eluted phages were then amplified in E. coli for the next round of biopanning. A total of 6 biopanning rounds have been performed independently on two anodic AI2O3 surfaces. After the 6th round, individual phage clones were sampled randomly from each surface to identify peptide sequences.

During successful biopanning experiments, phage binding yield should increase from one round to another round of selection enrichment [10]. Significant enrichment was indeed observed in our experiments on anodic AI₂O₃ during the first 3 biopanning rounds. During the next 3 biopanning rounds, the yield of phage binding stabilised, resulting in a final 400-fold increase. These results, both the increase in the initial stage and the stabilisation in the later stage, are already indicative for significant peptide affinity towards anodic alumina.

In order to identify the peptide sequences with specific affinity towards anodic alumina, DNA analysis has been carried after the sixth round. Peptide sequences of selectants randomly isolated from anodic AI2O3 surfaces are shown in Table 1. From the entire phage clone sample population, some very distinct features and consensus can be distinguished in the amino acid sequence, which is another indication of successful selection and of the specific affinity of some peptide sequences for anodic alumina surfaces.

The 6 rounds of panning procedures carried out on anodic alumina have allowed to isolate several phages that display peptides having identical sequences, like E-N-T-P-R-G-V-Q (7x), D-P-S-K-P-G-S-S (4x) and D-V-T-K-A-G-A-Q (2x). These selectant phage clones are grouped together in Table 1. To corroborate that these selectants indeed selectively bind to anodic AI2O3, 10¹² of each of the 3 isolated phages were grown up and the ability of the axemic phage lots to selectively bind to anodic AI₂O3 was verified by comparing to blanc experiments without any solid surface in solution. Ratio's in excess of 400 between the AI2O3 and blanc protocols were always obtained.

SEQ Table 1. Amino acid sequences for SEQ ID is

IDis successfully analysed selectant followed by inserted native phage clones. after native

SEQ ID No. 1 A D P A A R T Q V D P

SEQ ID No. 2 A E N T P R G V Q D P

SEQ ID No. 3 A D P S K P G S S D P

SEQ ID No. 4 A D P A K S P S S D P

SEQ ID No. 5 A D V T K A G A Q D P

SEQ ID No. 6 A D P G P K P S T D P

SEQ ID No. 7 A D P S L R N Q T D P

SEQ ID No. 8 A D T T K V P S Q D P

SEQ ID No. 9 A D V T K A T A Q D P

SEQ ID No. 10 A E P G K A S G S D P

SEQ ID No. 11 A E P G K G S A M D P

SEQ ID No. 12 A E P I K G G G S D P

SEQ ID No. 13 A E P P S G L L R D P

SEQ ID No. 14 A E P P S K A A M D P

SEQ ID No. 15 A E P S K A A G T D P

SEQ ID No. 16 A E P V Q K A G S D P

EXAMPLE 2; Phage binding to a Ta?θ5 surface using phage display technique

121

Anodic Ta₂θs surfaces were obtained by anodizing pure Ta thm films. A library of random peptides was used, containing 8 amino acids inserted in the pVIII coat protein of M13 bacteriophages in place of native EGD was screened using the biopanning protocol of Example 1. The sequences shown in table 2 were shown to exhibit selective and specific affinity to a Ta₂Os surface.

Table 2. Amino acid sequences for successfully analysed selectant phage clones (Ta₂O₅).

SEQ I D No . 17 D P A K G P A T

SEQ I D No . 18 D P A K S L G T

EXAMPLE 3: Biotinylation of the bacteriophages according to the invention does not prevent their binding to metal oxide surfaces

a) Bacteriophages of Example 1 modified with SEQ ID N0:3 have been purified

than been measured.

Phage purification by cesium chloride centrifugation

Equipment and reagents L8-M Beckman ultracentrifuge Swinging bucket SW40 rotor Cesium chloride (Sigma) TBS Purified phage preparation

Method

^■ Dilute the phage suspension with TBS to reach a final volume of 12 ml

^■ Add 5,4 g of cesium chloride and mix to dissolve

^■ Centrifuge the solution in a SW40 rotor for 17 h and 200000 g at 15°C

^■ Collect the fraction containing the phages ■ Dialyse the harvested solutions against TBS overnight in a float A Lyzer

G2 Phage modification with NHS-biotin

Purified phage preparation PEG 20%

Centrifuge Beckman, micro fuge 18 NaCl 100 mM/NaHCO₃ 10OmM buffer pH8 NHS-Biotin (Thermo scientific) TBS

Method ■ Add 1/5 volume of PEG in the 2 ml phage suspension

^■ Incubate for 1 h on ice

^■ Centrifuge 10 min at 10000 rpm (Centrifuge Beckman, micro fuge 18)

^■ Discard the supernatant and resuspend the pellet in 1,2 ml of buffer ( NaCl 100 mM/NaHCOs 100 mM pH 8) ■ Divide the volume in 6 aliquots of 200 μl

^■ Prepare samples of NHS-biotin at different final concentration (0,01 mM, 0,1 mM, 1 mM and 10 mM)

^■ Add 2 μl of NHS-biotin in each sample in order to obtain different final concentration (0 mM, 0,1 μM, lμM, 10 μM and 100 μm) ■ Incubate for lh30 at room temperature

^■ Add 1/5 volume of PEG and incubate for 15 min on ice

^■ Centrifuge 10 min at 10000 rpm (Centrifuge Beckman, micro fuge 18)

^■ Repeat the two last steps 3 times Resuspend the pellet in 200 μl of TBS

Adsorption on Al₂O₃ surface is then measured as described in Example 1. Results are presented in the table hereinafter:

b) Biotinylated bacteriophages prepared as described in a) have been tested for their ability to recognize streptavidine (ELISA protocol hereinafter).

ELISA

Equipment and reagents

24 wells plate (Nunclon Surface)

96 wells plate (VWR) 2% MPBS

TBS + 0,5% TWEEN 20

Streptavidin-Peroxidase Polymer, Ultrasensitive (Sigma)

3,3',5,5'-tetramethylbenzidine (TMB) liquid substrate system (Sigma T-8665)

H₂SO₄ IM Phage preparation

Method

^■ Place the AI2O3 surface in the wells of the 24 wells plate Wash the wells 10 times with 1 ml TBS IX + 0,5% TWEEN 20

^■ Add 50 μl of the phage suspension and 450 μl TBS IX + 0,5% TWEEN 20 to the wells ^■ Incubate for Ih at room temperature under shaking

^■ Discard the solution and wash the wells 5 times with 1 ml de TBS IX + 0,5% TWEEN 20 under shaking, 3 times with 1 ml PBS IX + 0,05% TWEEN 20 and 3 times withlml PBS IX ■ Add 400 μl/well HRP-streptavidin diluted 1 : 200 in 2% MPBS

^■ Incubate for Ih at room temperature

^■ Discard the solution and wash wells 3 times with TBS IX + 0,5% TWEEN 20 and 3 times with PBS Ix

^■ Add 400 μl TMB system solution to each well and incubate at room temperature for 10 min in the dark.

^■ Quench the reaction by adding 400 μl H₂SCU IM

^■ Read the plate at 450 nm

Results (see also Fig. 1)

As shown in figure 1, for a same concentration in streptavidin, when the concentration in biotin is increased, the recognition is not lost and signal is proportionally increased (col 1-5). Without alumina (col.6-9), there is no recognition. Col. 10-12 are controls. References

[1] Patwardhan S.V., Patwardhan G., Perry CC, J. Mater. Chem. 2007, 17,

2875

[2] Tamelrler C, Dincer S., Heidel D., Zareie M.H., Sarikaya M., Progr. Org. Coat. 2003, 47, 267

[3] Sano K. -L, Shiba K., J. Am. Chem. Soc. 2003, 125, 14234

[4] Willett R.L., Baldwin K. W., West K.W., Pfeiffer L.N., Proc. Natl. Acad.

ScL USA 2005, 102, 7817

[5] Willner L, Willner B., Katz E., Rev. Molec. Biotech. 2002, 82, 325 [6] Moreno -Hagelsieb L., Lobert P.E., Pampin R., Bourgeois D., Remacle J.,

Flandre D., Sensors and Actuators B 2004, 98, 269

[7] Zwick M.B., Shen J.Q., Scott J.K., Curr. Opin. Biotechnol. 1998, 9, 427 [8] Vanovermeere Q., Nysten B., Proost J., Appl. Phys. Lett. 2009, 94, 074103 [9] Soumillion P, Chapter 6 : Selection of phage-displayed enzymes, In: Evolutionary methods in biotechnology, clever tricks for directed evolution,

S. Brakmann & A. Schwienhorst ed., Weinheim, Germany, Wiley- VCH

Verlag GmbH, 2004, 47-64. [10] Merlin S., Rowold E., Abegg A., Berglund C, Klover J., Staten N et al,

Appl. Biochem. Biotechnol. 1997, 67, 199

Claims

1. A bacteriophage comprising a coat protein modified with at least one amino acid sequence, wherein said amino acid sequence comprises 4 to 17 amino acids, and wherein the modified coat protein is capable of binding to a metal oxide surface.

2. The bacteriophage according to claim 1, wherein said amino acid sequence comprises 5 to 12 amino acids, preferably about 8 amino acids.

3. The bacteriophage according to claim 1 or 2, wherein said coat protein is the pVIII coat protein and the bacteriophage is a filamentous bacteriophage.

4. The bacteriophage according to any one of claims 1 to 3, wherein said bacteriophage is a M 13, a fd or a fl mutant.

5. The bacteriophage according to any one of claims 1 to 4, wherein said amino acid sequence is selected from the group consisting of SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO:11, SEQ ID NO: 12, SEQ ID NO:13, SEQ ID NO: 14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO: 17 and SEQ ID NO:18.

6. The bacteriophage according to any one of claims 1-5, wherein said coat protein is the pVIII coat protein, and wherein said amino acid sequence is inserted in place of the amino acids EGD which are present in positions 2,3,4 of the amino acid sequence of the native pVIII coat protein, said amino acid sequence of the native pVIII coat protein being as shown in

SEQ ID NO: 19.

7. The bacteriophage according to any one of claims 1 to 6, wherein said bacteriophage further comprises a modified pill coat protein and/or a modified pIX coat protein, capable to bind to targets.

8. A bacteriophage comprising a modified pVIII coat protein, wherein said modified pVIII coat protein has an amino acid sequence selected from the group consisting of SEQ ID NO:20, SEQ ID N0:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID N0:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36 and SEQ ID NO:37.

9. The bacteriophage of claim 8, wherein said bacteriophage is a filamentous bacteriophage.

10. The bacteriophage of claim 9, wherein said bacteriophage is a M13, a fd or a fl mutant.

11. The bacteriophage according to any one of claims 8-10, wherein said bacteriophage further comprises a modified pill coat protein and/or a modified pIX coat protein.

12. The bacteriophage according to any one of claims 1-11, wherein said bacteriophage is genetically engineered.

13. The bacteriophage according to any one of claims 1-12, wherein said bacteriophage is biotinylated.

14. A nucleic acid sequence encoding an amino acid sequence selected from the group consisting of SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30,

SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36 and SEQ ID NO:37.

15. Biosensor comprising a bacteriophage as defined in anyone of claims 1 to 13.

16. Biosensor according to claim 15, further comprising a substrate at least partially coated with at least one metal oxide.

17. Biosensor according to claim 16, wherein the metal oxide is Al₂O₃, Ta₂O₅ and/or TiO₂.

18. Method for detecting and/or quantifying targets in a sample, comprising the following steps: - contacting a sample with a bacteriophage as defined in anyone of claims 1 to 13;

- said bacteriophage being or not being immobilized on a metal oxide surface,