WO2006008742A1

WO2006008742A1 - Novel sensors with nanoparticle probe

Info

Publication number: WO2006008742A1
Application number: PCT/IL2005/000775
Authority: WO
Inventors: Itamar Willner; Valeri Pavlov; Maya Zayatz; Tamara Niazov; Bella Shlyahovsky; Yi Xiao; Ronan BARON
Original assignee: Yissum Research Development Company Of The Hebrew University Of Jerusalem
Priority date: 2004-07-21
Filing date: 2005-07-21
Publication date: 2006-01-26

Abstract

The present invention concerns a method for the detection of the presence of an analyte in a sample, the analyte being one member of an enzyme/substrate pair, the method comprising: (a) providing nanoparticles of a transition metal, the other member of said pair; an electron acceptor and a transition metal salt capable to undergo a redox reaction with an electron donor thereby depositing a metal on said nanoparticles; (b) providing conditions enabling enzymatically catalyzed reaction between the substrate and the electron acceptor; (c) detecting a change in size of the nanoparticle; an enlargement of the nanoparticles' size indicating the presence of said one member in the sample.

Description

NOVEL SENSORS WITH NANOPARTICLE PROBE

FIELD OF THE INVENTION

The invention is generally in the field of sensory systems and relates to a nanoparticles functionalized probe and method for preparation thereof. The probe of the present invention is particularly useful in biosensor devices and assays.

LIST OF REFERENCES

The following references are considered to be pertinent for the purpose of understanding the background of the present invention:

1. J. Wang et al, J. Am. Chem. Soc. 2003, 125, 3214-3215;

2. J. Wang et al, Langmuir 2001, 17, 5739-5741.

3. I. Willner et al, Angew. Chem. Int. Ed. 2001, ¥0,1861-1864.

4. V. Pardo-Yissar et al, J. Am. Chem. Soc. 2003, 125, 622-623.

5. X.H. Gao, S.M. Nie, Trends Biotechnol 2003, 21, 371-373.

6. A.P. Alivisatos, Nat. Biotechnol. 2004, 22, Al -52.

7. LL. Medintz, et al, Nat. Mater. 2003, 2, 630-638.

8. T.A. Taton et al, Science 2000, 289, 1757-1760.

9. JJ. Storhoff et al, J. Am. Chem. Soc. 1998, 120, 1959-1964.

10. J. Liu, Y. Lu, J. Am. Chem. Soc. 2003, 125, 6642-6643.

11. L. He et al, J. Am. Chem. Soc. 2000, 122, 9071-9077.

12. F. Patolsky et al, Chem. Commun. 2000, 1025-1026; S. Han et al, Chem. Commun. 2001, 609-610.

13. I. Willner et al, Talanta 2002, 56, 847-856.

14. SJ. Park, et al, Science 2002, 295, 1503-1506.

15. A. Bardea et al, J. Am. Chem. Soc. 1997, 119, 9114-9119.

16. A. Doron et al, Langmuir 1995, 11, 1313-1317. 17. S. Link et al, J. Phys. Chem. B 1999, 103, 8410-8426.

The above references will be acknowledged in the text below by indicating their numbers [in brackets] from the above list.

BACKGROUND OF THE INVENTION

Increasing efforts are directed to the application of metal and semiconductor nanoparticles (NP) for the development of electronic or optical sensory systems. Metal or semiconductor NPs functionalized with nucleic acids were employed as amplifying labels for the detection of DNA. The dissolution of the nanoparticles was used to follow DNA hybridization events [1,2]. Also, charge injection from semiconductor nanoparticles into electrodes, and the generation of photocurrents was used to follow hybridization processes [3] and biocatalytic transformations [4].

The use of semiconductor quantum dots as fluorescence labels [5, 6] and the use of fluorescence quenching of semiconductor quantum dots in different sensing paths [7] are examples of sensory processes in the presence of metal and semiconductor NPs, where the detection is accomplished by optical means.

Au-nanoparticles (Au-NPs) were used as optical labels for the detection of biorecognition events such as DNA hybridization [8] or antigen-antibody complex formation [9] or for the analysis of the catalytic functions of nucleic acids [10]. Alternatively, Au-NPs conjugated to biomaterials were employed to amplify specific biomaterial binding events on surfaces by stimulating the electronic coupling between the localized plasmon of the NPs and the surface plasmon wave associated with the bulk Au-surface [H]. Also, Au-NPs were employed as "weight labels" for the detection of biorecognition events such as DNA hybridization using piezoelectric quartz crystals [12].

The intrinsic property of metal nanoparticles to catalyze the reduction of metal ions on the NPs and thereby to enlarge the metallic nanoparticles was employed in different biosensing paths. The catalytic enlargement of Au-NPs, acting as labels for DNA hybridization, was used for the amplified microgravimetric quartz-cry stal-microbalance detection of nucleic acids [13]. Similarly, the catalytic enlargement of Au-NP conjugates associated with biorecognition complexes was used to yield conductive patterns that follow biosensing processes [14].

SUMMARY OF THE INVENTION

The use of enzymes as biocatalysts for the enlargement of metallic particles is new. The present invention is based on the finding that enzymes, e.g. redox enzymes and hydrolases, can be used as biocatalysts for the electron- donor assisted enlargement or formation of metal nanoparticles. In a specific example, the enlargement of nanoparticles, e.g. gold, by reduction of gold salts and deposition of the metal gold formed on the existing nanoparticles, is achieved in the presence of the products of an enzymatically catalyzed reaction, e.g. H₂O₂, NAD(P)H, NADH, or Os(II) complexes. In another example, the generation of Au nanoparticles (also abbreviated hereinafter "Au- NP") by reduction of gold salts is used to detect enzyme e.g. tyrosinase, activity.

The enlargement or formation of the metal nanoparticles may be detected by optical, electrochemical, electrical, or microgravimetry means, and the detection is indicative of the presence an analyte in a sample, the analyte being one member of an enzyme-substrate pair.

Thus, the present invention concerns a method for the detection of the presence of an analyte in a sample, the analyte being one member of a pair enzyme/substrate, the method comprising:

(a) providing nanoparticles of a transition metal, the other member of said pair; an electron acceptor and a transition metal salt capable to undergo a redox reaction with an electron donor thereby depositing a metal on said nanoparticles;

(b) providing conditions enabling enzymatically catalyzed reaction between the substrate and the electron acceptor;

(c) detecting a change in size of the nanoparticle; an enlargement of the nanoparticles¹ size indicating the presence of said one member in the sample.

In a preferred embodiment, the present invention concerns a method for the detection of the presence of an analyte in a sample, the analyte being one member of a pair of oxidoreductase/oxidoreductase substrate, the method comprising:

(b) providing conditions enabling enzymatically catalyzed reaction between the oxidoreductase substrate and the electron acceptor;

(c) detecting a change in size of the nanoparticle; an enlargement of the nanoparticles' size indicating the presence of said one member in the sample.

In another preferred embodiment the analyte is a member of a pair of hydrolase/hydrolase substrate or a member of a pair of a hydroxylase/ hydroxylase substrate.

The method for the detection of the presence of the analyte as above, is a binary method, giving a yes/no answer. For example, if the enlargement of the nanoparticle size is beyond a threshold enlargement, (said threshold determined by measuring "spontaneous" enlargement in the absence of the analyte), this indicates the presence of the analyte (either the enzyme or the substrate) in the sample.

At times, it is desired not only to know whether the analyte (enzyme or its substrate) is present in the sample, but also to determine the amount of the analyte in the sample. In such a case, the present invention concerns a method for the determination of the amount of an analyte in a sample, the analyte being one member of a pair of an enzyme/ substrate, the method comprising:

(a) providing nanoparticles of a transition metal; the other member of said pair; an electron acceptor and a transition metal salt capable to undergo a redox reaction with an electron donor thereby depositing metal on said nanoparticles

(b) providing conditions enabling enzymatically catalyzed reaction between the enzyme substrate and the electron acceptor;

(c) detecting change in size of the nanoparticles;

(d) comparing the change in size of the nanoparticles with a calibration scale showing the relation between nanoparticles size change to the amount of the one member under said predetermined conditions; thereby determining the amount of said one member in the sample.

In a preferred embodiment the present invention provides a method for the determination of the amount of an analyte in a sample, the analyte being one member of a pair of an oxidoreductase/oxidoreductase substrate, the method comprising:

(c) detecting change in size of the nanoparticles;

The term "one member of a pair... " - refers to an analyte being either the substrate or the enzyme.

The term "the other member of said pair" refers, where the analyte to be detected is the enzyme, to the substrate, and, where the analyte to be detected is the substrate, to the enzyme. The term "oxidoreducta.se " refers to any enzyme which catalyzes an oxidation-reduction reaction, in the presence of an electron acceptor, producing an electron donor.

The electron acceptors of the oxidoreductase may belong to a wide group of compounds, but according to the present invention the preferred electron acceptors are oxygen (producing the electron donor of H₂O₂), NAD⁺/NAD(P)⁺ (resulting in the electron donors of NADH/NADPH and Os(III) complexes (producing the electron donor of Os(II).

Typically, the oxidoreductases are dehydrogenases, hydroxylases (i.e. enzymes which add -OH groups to its substrate during hydroxylation reactions by attaching oxygen atoms to it), reductases or oxidases as showed in a list bellow.

The term "oxidoreductase substrate " refers to any of the substrate on which the corresponding suitable enzyme works: for example the substrate of alcohol dehydrogenase is alcohol, the substrate of malate dehydrogenase is malate, the substrate of galactoseoxidase is galactose, the substrate of alcohol oxidase is alcohol etc.

The term "hydrolase" refers to any enzyme which catalyses the hydrolysis of various bonds. Systematic names of hydrolases are formed as "substrate hydrolase." However, common names are using only "substratease." For example, a nuclease is a hydrolase that cleaves nucleic acids. Within this group of enzymes are included esterases, deaminases, phosphotases, glycosylases, peptidases and the like.

The term "nanoparticles of a transition metal" refers to particles having a size in the nonometer size domain. The nanoparticles are having at least a transition metal outer coating. Although by a preferred embodiment the whole nanoparticle is composed of transition metal, it is possible that the nanoparticle has a "core and shell construct", wherein only the shell, on which the metal formed from the reduction of the metal salt is deposited, is made of the transition metal. The nanoparticles may be provided a priori in their final, metal reduced form, either in the solution conditions or on the solid substrate.

Alternatively the nanoparticles may be formed "in situ" either in the solution or on the substrate. In situ formation of the particles is assisted (but not mandatory) by providing "seeds" of the transition metal salts (which may be the same or different as the one used for the enlargement of the particles) together with an electron donor (such as CTAB or ascorbic acid) or a combination of electron donors (such as citrate and peroxide) in a basic pH, preferably pH about 10.

It has been surprisingly found that under the above conditions the nanoparticles can be produced in situ and subsequently enlarged in the presence of the analyte. In situ production of the nanoparticles is especially desirable where the electron acceptor is NAD⁺ , NADP⁺ or the like.

It should be noted that when the nanoparticles are produced "in situ", all the components of the reaction mixture needed to form enlargement (transition metal salt, electron donor/ combination of electron donors), together with electron acceptor, can be added at once, and all the reaction steps can take place under the same (basic) conditions.

Therefore, in the following wherever the term "nanoparticles" is used (as "provided" or "immobilized") this term refers also to "seeds" of a transition metal salt and to an electron donor/electron donor combination capable of forming nanoparticles of the transition metal under basic conditions.

The term "transition metal" is as defined by the periodic table, refers to elements in which filling of electrons in an inner d- or f-level occurs. The three main transition series that are typical metals, i.e. strong hard materials that are good conductors of heat and electricity and have high melting and boiling points, are as follows: from titanium to copper; from zirconium to silver; and from hafnium to gold.

The term "electron acceptor " in the context of the present invention refers to the moiety which accepts the electron resulting from the redox reaction between the oxidoreductase and its substrate. Typical examples are O₂, NAD⁺, NADP⁺, or transition metal complexes such as for example Os(III) complex (e.g. chloro-Os(III) bis-2,2'-bipyridine-mono-4-pyridine carboxylic acid dichloride), although this complex is merely a representative of a larger number of complexes possible. It should be understood that where the electron acceptor is oxygen, the term "providing" , mentioned in step (b) of the method, does not necessarily mean actively adding oxygen to the reaction mixture, as the basal amounts of oxygen may suffice for the reactions. In such a case, the term "providing ... electron acceptor" means enabling sufficient oxygen to penetrate the reaction mixture to produce the reaction.

The term "transition metal salt" refers typically to a salt of the transition metal, but may also refer to a transition metal complex that may be reduced to the metal. The transition metal salt should be such that it is capable of undergoing a redox reaction with an electron donor produced by the oxidoreductase substrate reaction. In the presence of the electron donor, the transition metal salt is converted into a metal that deposits on the nanoparticles.

Typically, the nanoparticle and the transition metal salt are of the same transition metal, preferably gold, however, there may be deposition of one transition metal (using one transition metal derivative) e.g. silver or copper on nanoparticles of another transition metal e.g. gold or vice- versa.

The terms "providing conditions enabling enzymatically catalyzed reaction ... " - refers to any conditions which are required for the production of the suitable electron donor as a result of the catalytic, enzyme-driven, reaction between the substrate and the electron acceptor. Such suitable conditions include temperature, pH, surfactant presence, electrolyte concentration.

Another important condition is the time period of the reaction, as enlargement is proportional to time of reaction and if calibration scale is used, it is important to assure that the time period for the reaction in the test is identical to the time period in which the calibration scale was produced

The conditions do not have to remain unchanged throughout the reaction, and it is possible that for the enzymatic reaction between the oxidoreductase and its substrate a first set of conditions is applied, and for the redox reaction between the electron donor and the transition metal salt which results in deposition on the nanoparticle, another set of conditions is applied. This is especially suitable where the electron donor is NADPH or NADH, wherein the first set of conditions for the enzymatic reaction should be a neutral pH, preferably in the range of pH 7-9 while the redox reaction that involves NAD(P)H itself should take place at an acidic pH, for example, pH of about 5 or below.

As a result of the electron donor - facilitated deposition of the transition metal on the nanoparticles, the size of the nanoparticles is changed, basically increased. The enlargement of the nanoparticles may be the indication of the presence of the analyte (enzyme or substrate in the sample) — or the amount of enlargement may be an indication of the amount of the analyte (enzyme/substrate) in the sample.

The detection of the change in size or generation of the nanoparticles may be achieved by various manners.

In a liquid solution the enlargement or generation of nanoparticles may be detected by optical means, such as by wavelength shift of electromagnetic irradiation or by change of absorbance of electromagnetic irradiation at one specific wavelength, the wavelength depending on the type of metal used.

In a specific example, the method of the present invention was also used for detecting tyrosinase activity since tyrosinase activity is involved in melanoma cells and Parkinson disease. In this case the formation of the nanoparticles in the system proceeds without added seeds. A series of neurotransmitters (L-DOPA, dopamine, adrenaline and noradrenaline) where used as the reducing agents for the generation of gold nanoparticles. The optical properties of the generated nanoparticles enable the quantitative analysis of the different neurotoransmitters, as well as that of the enzyme tyrosinase involved in this activity.

In another example, the method of the present invention was used for sensing nerve gases that act as acetylcholine esterase (abbreviated hereinafter "AChE") inhibitors. The inhibition of acetylcholine esterase by nerve gases, leads to the perturbation of the nerve conduction system and to the rapid paralysis of vital functions of the living systems. The AChE-mediated hydrolysis of acetylthiocholine yields a reducing agent thiocholine that stimulates the catalytic enlargement of Au NP seeds in the presence of AuCl^4". The reductive enlargement of the Au NPs is controlled by the concentration of acetylthiocholine and by the concentration of the enzyme. Consequently, the method of the present invention enables to follow after the inhibition of catalytic growth of the Au NPs by analyzing the acetylcholine esterase activity.

By a preferred embodiment of the present invention, the enzymatic reaction takes place on a solid substrate. This is achieved by immobilizing, in a manner as will be explained hereinbelow, the nanoparticles (or nanoparticle seeds) to the solid substrate. While the rest of the components of the method may be in solution, it is also possible to optionally immobilize the other member of the pair oxidoreductase/oxidoreductase substrate, on a solid substrate (especially if the member is the enzyme). Optionally, the electron acceptor can also be immobilized especially if the acceptor is NAD⁺ or NADP⁺. As used herein, the terms "immobilize" and "bind" and their derived meanings such as immobilized, immobilizing, bound, etc. denote chemical binding (i.e. chemisorption, covalent linkage e.g. hydrogen bonds, Van der Waals bonds, etc. or electrostatic linkage, e.g. ionic bonds, etc.) and physical binding.

The solid substrate may be selected from glass, conductive glass such as indium tin oxide (ITO), plastic polymer materials, ceramics, metal oxides, metals or any other solid material that enable the immobilization of nanoparticles/nanoparticles seeds. Where the solid substrate is an electrode the detection of the analyte is followed electrochemically by identifying the current or charge associated with electrical dissolution of the deposited matter.

Where the solid substrate is a non-conductive substrate separating two electrodes or wires, and the deposition of the metal occurs on the nanoparticles linked to the non- conductive domain, the detection of the enlargement is followed by identifying conductivity, resistance or capacitance changes between the electrodes or wires. Where the immobilization is on a piezoelectric quartz crystal, the detection may be by change of resonance frequency of the crystal, which is indicative of a change in the weight of the crystal. In general, this method is based on the principle that the oscillation of a crystal, both in frequency and amplitude, is in part a function of its weight. The change in weight of a crystal coated with a substrate selectively sensitive to a particular analyte when placed in an environment containing that analyte is, in turn, at least partly a function of the concentration of the analyte. Therefore, the measurement of the change in oscillation characteristics of a coated crystal sensitive to a particular analyte upon exposure to a given atmosphere is a direct and highly sensitive measure of the presence and concentration of that analyte.

The present invention also concerns a kit for the detection of the presence or the amount of an analyte in a sample, the analyte being one member of a pair of an oxidoreductase/oxidoreductase substrate comprising:

(a) nanoparticles of a transition metal; the other member of the pair; a transition metal salt capable to undergo a redox reaction with an electron donor thereby depositing metal on said nanoparticles and optionally an electron acceptor; and

(b) optionally a calibration scale showing the relation between an optical parameter and the amount of the one member.

Generally, the kit is also suitable for the detection of an analyte that is a member of any enzyme/enzyme substrate pair, for example a hydrolase/hydrolase substrate pair or hydroxylase/hydroxylase substrate pair.

The kit of the invention is suitable for detection of the analyte (enzyme or substrate) in a solution condition, without any immobilization on the solid substrate. Also, as explained above, the nanoparticles are either provided a priori or formed in situ from suitable reagents (e.g. seeds of transition metal salt and electron donor or combination of electron donors).

By another option, the present invention concerns a device for the detection of the presence or amount of an analyte in a sample, the analyte being one member of an enzyme/ substrate pair, the device comprising: a solid surface having immobilized thereon in a distinct location nanoparticles of a transitional metal.

Optionally the solid substrate may have immobilized thereon also the other member of the pair (especially where it is an enzyme), and optionally also the electron acceptors especially NAD⁺ NADP⁺,

The solid substrate may be glass, conductive glass such as indium tin oxide, plastic polymer materials, ceramics, metal oxides, metals or any other solid material that enable the immobilization of nanoparticles/nanoparticles seeds .

Where it is desired to carry out the invention using the device, the present invention also concerns a kit comprising a device having the nanoparticles, optionally also the other member of the pair immobilized on a solid substrate (possibly also with the electron acceptor), a transition metal salt, an electron acceptor (where the acceptor is other than oxygen which is naturally occurring in the ambient atmosphere and where the electron acceptor is not already immobilized on the substrate), the other member of the pair (where it is not immobilized on the substrate ) and optionally a calibration scale.

Where the substrate is for example glass, the calibration scale should be a calibration scale showing the relation between an optical parameter and the amount of the analyte to be detected ( "one member").

Where the solid surface is an electrode, a capacitor, a resistor or a non- conductive pathway connecting electrodes, the calibration scale should be a scale showing the relation between an electrochemical parameter (resistance, conductance, capacitance) and the amount of the analyte ( "one member").

Where the solid surface is a piezoelectric crystal, the calibration scale should show the relationship between a microgravity parameter such as resonance frequency, and the amount of the analyte { "one member").

The term "calibration scale " refers by one embodiment to a graph showing in its "X" axis varying amounts of the analyte to be detected (either the enzyme or the substrate) and at its "Y" axis the level of the parameter which is to be tested (optically - wavelength shift or new absorbance; electrochemically — resistance, capacitance or conductivity, microgravity — weight) wherein the determination of the parameter was undertaken under the same conditions as those of the assay of the present invention. By another option the scale may be for example a table of color codes to which the colorimetric reaction should be compared.

GENERAL DESCRIPTION OF THE INVENTION

Oxidoreductase/oxidoreductase substrates

As described above, preferably the enzymes are oxidoreductases, hydroxylases or hydrolases (e.g. acetylcholine esterase). Oxidoreductases should be of the type wherein the electron acceptor is oxygen, NAD⁺/NADP⁺ or transition metal complexes such as Os (III) complex.

Examples of enzymes wherein their natural electron acceptor is either oxygen or NAD⁺TNADP⁺ are given bellow. The suitable substrate can be determined by looking at the first part of the enzyme.

Transitional metal complexes, such as Os-complexes include the transition metal linked to appropriate ligands.

Preferred analytes in accordance with the invention are glucose, lactose, alcohol, cholesterol , choline, choline-esterase inhibitors.

Oxidoreductases

With NAD or NADP as acceptor alcohol dehydrogenase alcohol dehydrogenase (NADP) homoserine dehydrogenase (R,R)~butanediol dehydrogenase glycerol-3 -phosphate dehydrogenase (NAD) D-xylulose reductase L-iditol 2-dehydrogenase mannitol- 1 -phosphate 5-dehydrogenase UDP-glucose 6-dehydrogenase histidinol dehydrogenase quinate 5 -dehydrogenase shikimate 5 -dehydrogenase L-lactate dehydrogenase D-lactate dehydrogenase 3 -hydroxy isobutyrate dehydrogenase hydroxymethylglutaryl-CoA reductase (NADPH2) 3-hydroxyacyl-CoA dehydrogenase acetoacetyl-CoA reductase malate dehydrogenase malate dehydrogenase (oxaloacetate-decarboxylating) malate dehydrogenase (oxaloacetate-decarboxylating) (NADP) isocitrate dehydrogenase (NAD) isocitrate dehydrogenase (NADP) phosphogluconate dehydrogenase (decarboxylating) glucose 1 -dehydrogenase glucose-6-phosphate 1 -dehydrogenase fructuronate reductase -hydroxybutyrate dehydrogenase estradiol 17b-dehydrogenase pyridoxine 4-dehydrogenase gluconate 5 -dehydrogenase octanol dehydrogenase D-malate dehydrogenase (decarboxylating) 3-isopropylmalate dehydrogenase ketol-acid reductoisomerase hydroxymethylglutaryl-CoA reductase aryl-alcohol dehydrogenase aryl-alcohol dehydrogenase (NADP) tartrate dehydrogenase phosphoglycerate dehydrogenase 3 -oxoacyl- [acyl-carrier-protein] reductase 2-deoxy-D-gluconate 3 -dehydrogenase 15-hydroxyprostaglandin dehydrogenase (NAD) ureidoglycolate dehydrogenase 3-hydroxybutyryl-CoA dehydrogenase D-xylose 1 -dehydrogenase (NADP) carbonyl reductase (NADPH2) cinnamyl-alcohol dehydrogenase xanthine dehydrogenase IMP dehydrogenase gluconate 2-dehydrogenase dihydrokaempferol 4-reductase tropinone reductase methanol dehydrogenase formaldehyde dehydrogenase (glutathione) formate dehydrogenase aldehyde dehydrogenase (NAD) aldehyde dehydrogenase (NADP) aldehyde dehydrogenase [NAD(P)] betaine-aldehyde dehydrogenase aspartate-semialdehyde dehydrogenase glyceraldehyde-3 -phosphate dehydrogenase (phosphorylating) succinate-semialdehyde dehydrogenase [NAD(P)] aminobutyraldehyde dehydrogenase methylmalonate-semialdehyde dehydrogenase (acylating) L-aminoadipate-semialdehyde dehydrogenase N-acetyl-g-glutamyl-phosphate reductase phenylacetaldehyde dehydrogenase glutamate-5-semialdehyde dehydrogenase formaldehyde dehydrogenase enoyl-[acyl-carrier-protein] reductase (NADH2) prephenate dehydrogenase (NADP) orotate reductase (NADH2) trans- 1 ,2-dihydrobenzene- 1 ,2-diol dehydrogenase 2,4-dienoyl-CoA reductase (NADPH2) 12-oxophytodienoate reductase 2'-hydroxyisoflavone reductase glutamate dehydrogenase glutamate dehydrogenase (NADP) glutamate synthase (NADPH2) glutamate synthase (NADH2) pyrroline-5-carboxylate reductase dihydrofolate reductase methylenetetrahydrofolate dehydrogenase (NADP) saccharopine dehydrogenase (NAD, L-lysine-forming) saccharopine dehydrogenase (NADP, L-glutamate-forming)

1 -pyrroline-5-carboxylate dehydrogenase methylenetetrahydrofolate dehydrogenase (NAD)

D-nopaline dehydrogenase methylenetetrahydrofolate reductase (NADPH2)

NAD(P) transhydrogenase (B-specific)

With a heme protein as acceptor cytochrome-b5 reductase

NADPH — ferrihemoprotein reductase sulfite reductase (NADPH2) dihydrolipoamide dehydrogenase mercury(II) reductase ferredoxin — NADP reductase

With oxygen as acceptor dihydroorotate oxidase coproporphyrinogen oxidase protoporphyrinogen oxidase acyl-CoA oxidase L-gulonolactone oxidase galactose oxidase alcohol oxidase (S)-2-hydroxy-acid oxidase xanthine oxidase L-galactonolactone oxidase cellobiose oxidase D-aspartate oxidase L-amino-acid oxidase D-amino-acid oxidase amine oxidase (flavin-containing) pyridoxamine-phosphate oxidase amine oxidase (copper-containing) putrescine oxidase sarcosine oxidase polyamine oxidase urate oxidase sulfite oxidase cytochrome-c oxidase catechol oxidase laccase

L-ascorbate oxidase glucose oxidase lactate- oxidase choline- oxidase alcholol-oxidase cholesterol-oxidase xanthine oxidase pyridoxol oxidase hexose oxidase L-sorbose oxidase

SAMPLE

The sample may be a priori, in a liquid sample such as a body fluid (saliva, blood, plasma, sperm, urine, cerebro-spinal fluid, etc.), various waste products such as sewage water, fluid obtained from industrial reactions, e.g. in the food (juices, wines), cosmetic, agricultural (fruits, milk) or pharmaceutical industry (to test amount of substrate or enzyme used or produced in these reactions), liquid solutions used for laboratory and research purposes (to test reagents, starting materials, enzymes or products of these reactions), liquid or gases (such as nerve-gas) used in biological or chemical ware fare for homeland security etc. Alternatively, the sample may be gas, such as for example for the detection of alcohol in the breath of a person suspected of driving drunk, detection of biological or chemical warfare agents (such as nerve gas) in the atmosphere etc.

Immobilization on a Solid Substrate

When the method of the invention is carried out on a solid substrate, the metal nanoparticles are immobilized on the substrate. Such immobilization may be carried out by modifying the surface of the substrate so as to enable the association to the particles. In the case of glass surfaces for example, Au colloid films on glass surfaces were prepared by modification of the surfaces with linker molecules. Suitable linker molecules are organic molecules having at least two functional groups, one of the functional groups being capable to react and bind to the substrate surface and another of the functional groups being capable to react and bind to the nanoparticles. Non-limiting examples of suitable functional groups are silane, thiols, carboxylate, amines and the like.

Preferred organosilanes are those bearing a functional group X that may vary widely depending on the desired properties. By way of example and not limitation, X may be COOH, OH, NH(R), N(R)₂, NH₂, COOR, where R represents organic functional groups in general, e.g. hydrocarbon or halocarbons. Non-limiting examples of organosilanes that may be used for primary modification of the surfaces are aminopropyl triethoxysilane, aminopropylmethyldiethoxysilane, aminoethylaminopropylmethyldimethoxy- silane, diethylenediaminopropyl-trimethoxysilane, cyclohexylaminopropyl- trimethoxysilane, anilinomethyltriethoxysilane, methacryloxypropyltriethoxy- silane, chloromethyltriethoxysilane, mercaptopropyltrimethoxysilane and the like. In a specific example, schematically showed in Figure 1, Au colloid films on glass surfaces were prepared by primary modification of the surfaces with (aminopropy 1 )siloxane.

When the surfaces are made of a metal or coated with a metal such as for example gold, silver, platinum, iridium, cobalt, chromium or alloys or mixtures such as Co-Cr, Cr-Au, Pt-Ir, and Ti-Pt, the bifunctional ligands to be used should have high affinity to metal surfaces. Examples include thiol functionality that binds strongly to gold, silver and platinum surfaces, e.g. dithiols such as hexane- dithiols, aminothiols and the like.

Optical

Where the reaction is detected optically, the device may have the shape of a simple probe used in various colorimetric reactions (glucose test, pregnancy tests ,etc) such as a stick (made of paper, cardboard, comprising a membrane etc) to be dipped in the sample. Alternatively the device may be an apparatus, such as use for home testing of pregnancy, having a paper or membrane bearing the immobilized nanoparticles sandwiched between two plastic surfaces forming together a receptacle, (where the surface-bearing the particles is exposed) capable of receiving a certain amount of the liquid sample etc.

If the optical detection is by wavelength shift or change of wavelength, the new color produced may be indicative to the presence of the analyte in the sample. Where the optical detection is by absorbance, preferably the solid surface should be transparent, and the detection of the device may be achieved using an external system (photometer) for checking change in absorbance.

In order to verify that all components of the device are in working order it is preferable to provide a test zone (having immobilized nanoparticles, the other member of the pair and optionally the electron acceptor) which gives an optical indication only in the presence of the analyte and a control zone (comprising in addition also the member to be detected - i.e. comprising the analyte)- to produce a positive control.

Electrochemical /Electrical

Where the device is used for electrochemical testing, an electrochemical cell is provided, comprising an electrolyte solution, a working electrode, a counter electrode, and a reference electrode. By application of an appropriate potential on the working electrode relative to the other electrodes a redox- reaction dissolving the deposited metal occurs. The current or charge associated with the dissolution will than be monitored to determine the amount of the deposited matter.

The electrical measurement of the enlarged particles on the non- conductive surface separating two conductive wires (or electrodes) may monitor, in solution or in dry state, changes in parameters which occur as a result of the enlargement of the particles such as changes in conductivity, resistance, or capacitance, Piezoelectric

In general, this method is based on the principle that the oscillation of a crystal, both in frequency and amplitude, is in part a function of its weight. Where the detection is by piezoelectric means, a crystal element should contain at least one crystal resonator having a membrane-like region and being characterized by a certain resonance frequency value, a surface region of said at least one crystal resonator being modified by reactive molecules of a kind capable of interacting with at least one analyte material to yield a reaction product that effects a change in the resonance frequency of said crystal resonator from said certain resonance frequency value, said change being indicative of the identity and quantity of said at least one foreign material.

BRIEF DESCRIPTION OF THE DRAWINGS

In order to understand the invention and to see how it may be carried out in practice, a preferred embodiment will now be described, by way of non- limiting example only, with reference to the accompanying drawings, in which:

Figure 1 is a scheme showing the assembly of Au-NPs on a glass substrate and the biocatalytic Au-NPs growth in the presence of glucose. Figure 2A is a graph showing the absorbance spectra of Au-NP seeds (12 ± 1 nm), 3 x 10^'10 M, upon growth in the presence Of HAuCl₄, 2 x 10^"4 M, in 0.01 M phosphate buffer solution that included CTAC, 2 x 10^'3 M, and using different concentrations Of H₂O₂: a) O M, b) 2 x 10^'5 M, c) 5 x 10^"5 M, d) 1.1 x 10^"4 M, e) 1.8 x 10^'4 M, f) 2.4 x 10^"4 M, g) 6.0 x 10^"4 M. All spectra were recorded after a reaction time interval of 5 minutes.

Figure 2B is a calibration scale corresponding to the absorbance at λ = 529 nm of the enhanced Au-NPs solution at variable concentrations of H₂O₂. (Experimental conditions as in Fig. 2A)

Figure 3 shows HR-TEM images of: 3A) The Au-NP seeds; 3B) after 5 min. of reaction, with HAuCl₄, 2 x 10^"4 M, CTAC, 2 x 10^"3 M, and 5 x 10^"5 M H₂O₂; 3C) and 3D) after reaction with HauCl₄, 2 x 10^"4 M, CTAC, 2 x 10^'3 M, and 2.4 x 10^"4 M H₂O₂.

Figure 4 shows a Fourier filtered HR-TEM image of a 3.5 nm detached crystalline cluster. Lattice fringe of the 0.235 nm of { 111 } Au crystal plane is indicated.

Figure 5 shows HR-TEM image of Au-NP and 3.5 nm height triangular shape flake (marked with arrow) grown on its surface. Inset: Fast Fourier Transformed image of the flake area confirming its orientation along <0 I n direction.

Figure 6 shows HR-TEM image of an enlarged Au-NP with nanocrystalline clusters at faces intersections.

Figure 7A is a graph showing the absorbance spectra of the Au-NP- functionalized glass surfaces after 10 min of reaction with 2 x 10^" M HAuCl₄ in 0.01 M phosphate buffer solution that includes CTAC upon reaction with different concentrations Of H₂O₂: a) 0 M₅ b) 2 x 10^"5 M, c) 5 x 10^"5 M, d) 8 x 10^"5 M, e) 2.4 x lO^"4 M, f) 6.0 x 10^"4 M, g) 7.2 x 10^"3 M.

Figure 7B is a calibration scale corresponding to the absorbance changes (at λ = 535 nm) of the Au-NP- functionalized glass supports upon enlargement in the presence of different concentrations of H₂O₂. (Experimental conditions as in 7A)

Figure 8 show HR-SEM images of Au-NP-functionalized ITO glass slides: 8A) after preparation 8B) after 10 min of reaction with HAuCl₄, 2 x 10^"4 M, in 0.01 M phosphate buffer and CTAC, 2 x 10^"3 M ,and H₂O₂, 1.8 x 10^"4 M.

Figure 9A is a graph showing the absorbance spectra of solutions containing Au-NP seeds, 3 x lO^'10 M, 2 x 10^"4 M HAuCl₄, 47 μg.ml^'1 GOx, in 0.01 M phosphate buffer solution and CTAC, 2 x 10^"3 M, upon reaction for 10 min at 30⁰C, with different concentrations of β-D(+) glucose: a) 0 M, b) 2 x 10^" ⁶ M, c) 1 x 10^"5 M, d) 2 x 10^"5 M, e) 5 x 10^"5 M, f) 1.1 x 10^"4 M, g) 2.4 x 10^"4 M.

Figure 9B is a calibration scale corresponding to the absorbance at λ = 535 nm of the enhanced Au-NPs solutions upon interaction with different concentration of β-D(+) glucose. (Experimental conditions as in 9A)

Figure 1OA is a graph showing the absorbance spectra of the Au-NP- functionalized glass supports upon reaction with 2 x 10^"4 M HAuCl₄, 47μg.ml^"1 GOx in 0.01 M phosphate buffer that includes CTAC, 2 x 10^"3 M, and different concentrations of β-D(+) glucose: a) 0 M, b) 5 x 10^"6 M, c) 2 x 10^"5 M, d) 5 x 10^"5 M, e) 1.1 x 10^"4 M, f) 1.8 x 10^"4 M, g) 3.0 x 10^"4M. For all experiments the reaction time interval was 10 minutes, 3O⁰C.

Figure 1OB is a calibration scale corresponding to the absorbance at λ = 542 nm of the Au-NP-functionalized glass supports upon analyzing variable concentrations of β-D(+) glucose. (Experimental conditions as in 10A)

Figure HA is a graph showing the spectral changes of the Au-NP seed solution, 4.0 x 10^"10 M, that includes HAuCl₄, 1.8 x 10^"4 M, and CTAB, 7.4 x 10^"2 M, upon the addition of different concentrations of NADH: (a) 0 M; (b) 4.2 x 10^"5 M; (c) 8.4 x 10^"5 M; (d) 12.6 x 10^"5 M; (e) 21 x 10^"5 M; (f) 30 x 10^"5 M; (g) 42 x 10^"5 M; (h) 63 x lO^'5 M. Inset: Calibration scale corresponding to the spectral changes of. the growth solution upon interaction with variable concentrations of NADH (30 minutes). Absorbance recorded at λ = 524 nm. Figure HB is graph showing the spectral changes of the Au-NP seed solution, 4.0 x 10^"10 M, that includes HAuCl₄, 1.8 x 10^"4 M, and CTAB, 7.4 x 10^"2 M, upon the addition of different concentrations of NADPH: (a) 0 M; (b) 1.79 x 1(T⁵ M; (c) 3.57 x 1(T⁵ M; (d) 10.7 x IO^{^5} M; (e) 18 x 10^"5 M; (f) 25 x 10^" ⁵ M; (g) 36 x 10^'5 M; (h) 54 x 10^"5 M; (i) 71 x 10^"5 M. Inset: Calibration scale corresponding to the spectral changes of the growth solution after interaction with variable concentrations of NADPH (30 minutes). Absorbance recorded at λ = 524 nm.

Figure 12 is a graph showing the absorbance changes of the Au-NP- aminopropylsiloxane-functionalized glass slides upon interaction with HAuCl₄,

1.8 x 10^"4 M, and CTAB, 7.4 x 10^"2 M, in the presence of different concentrations of NADH: (a) 1.36 x IO^{^3} M; (b) 68 x 10^"5 M; (c) 65 x 10^"5 M; (d) 61 x 10^"5 M; (e) 58 x 10^"5 M; (f) 54 x 10^"5 M; (g) 51 x 10^"5 M; (h) 48 x 1O^-5 M; (i) 44 x 10^"5 M; G) 41 x 10^"5 M; (k) 34 x 10^"5 M; (1) 27 x 10^"5 M; (m) 14 x 10-⁵ M; (n) 0 M.

Figure 13 shows SEM images of enlarged Au-particles generated on a Au-nanoparticle-3-aminopropylsioloxane interface on a glass support using HAuCl₄ ^", 1.8 x 10-⁴ M, CTAB, 7.4 x 10^"2 M and NADH: (A) 6.8 x 10^"5 M; (B) 27 x 10-⁵ M; (C) 41 x 10^"5 M; (D) 54 x 10^'5 M; (E) 61 x 10^"5 M; (F) 1.36 x 10^'3 M. For all images the scale bar is equal and corresponds to 200 nm. All surfaces are coated with a Au/Pt layer (7-8 nm) to enhance the conductivity of the supports.

Figure 14A is a graph showing the spectral changes of the growth solution that includes Au-NP seeds, 1.4 x 10^"10 M, HAuCl₄, 1.8 x 10^"4 M, CTAB, 7.4 x 10^"2 M, upon the addition of biocatalytically generated NADH in compartments that include LDH, 0.2 mg mL^"1, NAD⁺, 1 x 10^"3 M, and variable concentrations of lactate (time-interval for the biocatalytic transformation 30 minutes): (a) 0 M; (b) 2.3 x 10^"3 M; (c) 2.9 x 10^"3 M; (d) 3.4 x 10^'3 M; (e) 4.0 x 10^"3 M; (f) 4.6 x 10^"3 M; (g) 5.2 x 10^'3 M; (h) 5.7 x 10^"3 M; (i) 6.3 x 10^"3 M; (j)

6.9 x 10^"3 M; (k) 9.2 x 10^"3 M. Inset: Calibration scale corresponding to the spectral changes of the growth solution as a function of lactate concentration in the biocatalyzed transformation compartment.

Figure 14B is a graph showing the spectral changes of the Au-NP-3- aminopropylsiloxane-functionalized glass slides upon interaction with the growth solution consisting Of HAuCl₄, 1.8 x 10^"4 M₅ CTAB, 7.4 x 10^"2 M; and the interaction with biocatalytically-generated NADH formed within 30 minutes in compartments that included LDH, 0.2 mg mL^"1, NAD⁺, 1 x 10^'3 M, and variable concentrations of lactate: (a) 9.8 x 10^"3 M; (b) 7.3 x 10^"3 M; (c) 6.6 x 10^"3 M; (d) 5.8 x 10^"3 M; (e) 5.1 x 10^"3 M; (f) 3.6 x 10^"3 M; (g) 2.9 x 10^"3 M; (h) 0 M.

Figure 15 shows a UV- vis. absorbance spectra of a solution initially containing 3 x 10^"10 M 12 nm Ag-NPs, 2 x 10^"4 M HAuCl₄, 2 x 10^'4 M HAuCl₄ in 0.01 M phosphate buffer and 2 x 10^"3 M CTAC after reaction, RT 5 min: a) without H₂O₂ b) with 1.1 x 10^'4 M H₂O₂. The spectra were recorded after a reaction time interval of 5 minutes

Figure 16 is graph showing the spectral changes of the Au-NP seed solution, 4.0 x 10^"11 M, that includes HAuCl₄, 1.5 x 10^"3 M, and Osmium complex (chloro-Os(III) bis-2,2'-bipyridine-mono-4-pyridine carboxylic acid dichloride, also abbreviated Os(III)(bpy)₂(py-CO₂H)Cl]Cl₂) 1 x 10^"4 M, citrate 7.5 x 10^"3 M and GOx upon the addition of different concentrations of glucose: (a) 0 M; (b) 7.4 x 10^"5 M; (c) 3. 7 x 10^"4 M; (d) 7.4 x 10^"4 M; (e) 1.48 x 1O^-3 M; (f) 2.95 x 10-³ M; (g) 4.43 x 10^"3 M; (h) 5.9 x 10^"3 M; (i) 71 x 10^"5 M. Absorbance recorded from 360 nm to 900 nm.

Figure 17 is a graph showing spectral changes of the generated Au-NP produced in the solution that includes HAuCl₄, 7.9 x 10^"4 M, H₂O₂, 0.027 M and citrate, 7.9 x 10^"3 M, upon the addition of different concentrations of NADH: (a) 0 M; (b) 1.1 x 10^"6 M; (c) 3.4 x 10^"6 M; (d) 6.7 x 10^"6 M; (e) 1.1 x 10^"5 M; (f) 5.6 x 10^"5 M; (g) 7.8 x 10^"5 M (under air). Absorbance recorded from 400 nm to 900 nm. Figure 18 is a graph showing spectral changes of the generated Au-NP solution produced in the solution that includes HAuCl₄, 7.9 x 10^"4 M, H₂O₂, 0.027 M, citrate, 7.9 x 10^"3 M, NAD⁺, 1 x 10^"3 M, and glucose dehydrogenase, 0.3 mgmL^"1, upon the addition of different concentrations of glucose: (a) 0 M; (b) 6 mM (under air). Absorbance recorded from 400 nm to 900 nm.

Figure 19 is a graph showing absorbance spectra of Au-NPs formed by variable concentrations of tyrosinase: a) 0, b) 10 U ml^"1, c) 20 U ml^"1, d) 30 U ml^"1, e) 35 U ml^"1, f) 40 U ml^"1, g) 60 U ml^"1. All systems include tyrosine, 2 x 10^"4 M, HAuCl₄, 2 x 10^"4 M, CTAC, 2 x 10^"3 M, in 0.01 M PB. Spectra were recorded after a fixed time-interval of 10 minutes. Inset: Calibration curve corresponding to the absorbance at λ = 520 nm of the Au-NPs formed in the presence of variable concentration of tyrosinase.

Figure 20 schematically shows the detection of acetylcholine esterase activity by growing Au nanoparticles.

Figure 21 is a graph showing the absorbance spectra corresponding to the inhibition of the Au NPs growth on glass supports recorded in the presence ofAChE, 0.13 units/mL, HAuCl₄, l.lxlO^"3 M₅ [acetylthiocholine] = 1.4x10^" ⁴M, and different concentrations of inhibitor: (a) 0 M; (b) 5.9xlO^"7 M; (c) 1.2xlO^"6M; (d) 2.4xlO^"6 M; (e) 5.9xlO^"6 M.

DETAILED DESCRIPTION OF THE INVENTION

The present invention will be described by the following non-limiting examples:

A. Catalytic Growth of Au-NPs Using AuCl₄ ^' and H₂O₂

The method and device of the invention are exemplified through a glucose sensor system based on the biocatalytic enlargement of Au-NPs. The glucose system may provide a model for numerous other oxidase-based biosensor assemblies.

The addition OfH₂O₂ to a 0.01 M phosphate buffer solution that includes AuCl₄ ^", 2 x 10^"4 M, Au-NP seeds (12 ± 1 nm stabilized by citrate), 3 x 10^"10 M, cetyltrimethylammonium chloride (CTAC), 1 x 10^"3 M, as surfactant, results in the immediate increase of the absorbance corresponding to the Au-NPs plasmon. CTAC is a non limiting example of a suitable class of surfactants which may be used in the method of the present invention. Other examples of such surfactants are quaternary ammonium or phosphonium salts.

Figure 2A shows the evolution of the absorbance spectra in the system in the presence of variable concentrations of H₂O₂ (spectra recorded after a fixed time-interval of 5 min). As the concentration of H₂O₂ increases the absorbance is higher, and Figure 2B shows the derived calibration scale. Control experiments reveal that no Au-NPs are formed in the absence of the Au-NP seeds and that added H₂O₂ is essential to stimulate the absorbance changes. These results suggest that the Au-NP seeds act as catalysts for the reduction of AuCl₄ ^" by H₂O₂ resulting in the enlargement of the particles and the enhanced absorbance features.

A closer inspection of the spectral changes of the Au-NP seeds upon treatment with H₂O_{2 )} Figure 2A indicates that at low H₂O₂ concentration a red shift (ca. 15 nm) in the absorbance maximum is observed, scale (a), while at higher H₂O₂ concentrations, concomitant to the absorbance, growth a blue-shift in the absorbance maxima (ca. 10 nm as compared to the seeds). While the red- shift observed at low H₂O₂ concentrations may support the enlargement of the particles, the blue-shift at higher H₂O₂ concentrations with the concomitant absorbance growth may imply that in solution there is formation of more Au- NPs of lower dimensions or the formation of a mixture of very small crystalline Au-NPs together with enlarged Au-NPs. To understand the mechanism of growth of the Au-NPs the inventors have performed a detailed HR-TEM analysis that follows the enlargement process: Figure 3 A shows the HR-TEM image of the Au-NP seeds. From this figure it is seen that the Au-NPs exist in several morphologies (spheres, rhombs, triangles and polygons) with a very narrow size distribution, 12 ± 1 nm. Figures 3B - 3D show the NPs formed upon treatment of the Au-NP seeds with a low (5 x 10^"5 M) and a high (2.5 x 10^"4 M) concentration of H₂O₂ in the presence OfAuCl₄VCTAC (for 5 minutes), respectively. For the low concentration Of H₂O₂, Figure 3B, there are observed NPs with a strong dark contrast of dimensions corresponding to ca. 13 x 13 nm that are coated by numerous nanocrystallites of lighter contrast to yield Au-NP clusters of dimensions up to ca. 18 x 27 nm. For the higher concentration of H₂O_2, Figure 3 C and 3D, the Au-NPs coated with the Au-nanocrystallites reached larger dimensions, up to ca. 32 x 28 nm, but in addition to these enlarged particles numerous small separated Au-nanocrystal flakes of variable sizes, 2.5 to 7nm (with maximum distribution of 3.5 nm) are observed. Figure 3C shows separated clusters marked with arrows.

Figure 4 shows the Fourier filtered image of a 3.5 nm sized separated flake in which lattice fringes corresponding to the (111) plane of Au⁰ are clearly resolved. The majority of the observed flakes contain dislocations and part of them were found to be folded and to appear as two-dimensional Au- crystallites. Figure 5 shows the HR-TEM image of the Au-NP that includes the attached, catalytically-grown, Au nanocrystals on the core Au-NP seed. It was observed that the Au-crystallites are catalytically grown at the intersection of the faces of the parent seed Au-NPs. The HR-TEM analysis indicates that the initial Au-NP seeds are in the distinct decahedral or icosahedral structures. Upon the enlargement of the particles, Figure 6, gold is deposited on the seeds and the nanocrystalline flakes are deposited and catalytically enlarged at the sharp intersections of (111)/(111), (100)/(110) and (110)/(210) faces. This mechanism may smooth out the sharp edges of the NPs and decrease their surface energy.

The enlargement of the Au-NPs seeds by H₂O₂ was also examined on solid surfaces. The citrate-capped Au-NPs were assembled on an aminopropylsiloxane film that was assembled on glass plates. The resulting plates were then reacted with solution of AUCI₄VCTAC in the presence of different concentrations of H₂O₂. The absorbance of the surfaces was checked in water after 10 min of reaction. Figure 7A shows the absorbance spectra of the Au-NP modified surfaces upon treatment with different concentrations of H₂O₂, where Figure 7B depicts the derived calibration scale. The absorbance of the Au-NP modified interface increases as the concentration of H₂O₂ is elevated. In contrast to the spectra in solution, the absorbance bands characteristic to the Au-NPs are constantly red-shifted. As the H₂O₂ concentration increases the Au-NPs are enlarged, but the small flakes separated from the parent Au-NP cores are washed off into the growing solution. The absorbance spectra of the slides correspond then to the net surface enlarged Au- NPs. HR-SEM measurements support the enlargement process of the Au-NPs on the surface. The HR-SEM images of the Au-NP seeds deposited on aminopropylsiloxane-functionalized ITO plates prior to reaction with H₂O₂ and after reaction with H₂O₂, 1.8 x 10^"4 M, for 10 minutes are shown in Figure 8 A and 8B respectively. The Au-NPs are enlarged from 12 ± 1 nm to 18 ± 1 nm, respectively. The size of the enlarged particles elucidated by the HR-SEM measurements is in very good agreement with the size of the particles deduced from the respective absorbance spectrum and is in good agreement with the theoretically calculated value using the Mie theory [17].

Numerous oxidases generate H₂O₂ upon the oxidation of the respective substrates by molecular O₂. Therefore, according to the present invention, the catalytic growth of Au-NPs is employed as a process for the optical sensing of a respective substrates. In a method for the optical detection of glucose, glucose oxidase, GOx, and O₂/glucose were used as the H₂O₂ generating system. Figure 9A shows the spectral changes of the Au-NP seeds solution, 3 x 10^"10 M, that included AUCI₄VCTAC and GOx, 47 μg ml^"1, upon the addition of different concentrations of glucose (the experiments were performed under O₂ at 30⁰C and the spectra were recorded after a fixed time- interval corresponding to 5 min). The absorbance spectra of the particles increase as the concentration of glucose is elevated. The calibration scale is depicted in Figure 9B. Control experiments indicate that no growth of Au-NPs occurs upon exclusion of glucose, GOx, or in the absence of O₂. These results indicate that the biocatalyzed formation of H₂O₂ is essential to induce the growth of the particles. As the concentration of glucose increases the concentration of the generated H₂O₂ is higher and the growth of the Au-NPs is enhanced.

The analysis of the glucose by this method was also performed on glass supports, as schematically shown in Figure 1. Au-NP seeds were immobilized to aminopropylsiloxane that was bound to a glass surface and the resulting functionalized support was interacted with a solution of AuCl₄7CTAC/GOx in the presence of variable concentrations of glucose for 10 min at 30⁰C. Figure 1OA shows the spectral changes of the glass slides upon the growth of Au-NPs in the presence of variable concentrations of glucose. Figure 1OB shows the derived calibration scale corresponding to the optical detection of glucose by the catalytically enlarged Au-NPs. The increase of the absorbance values as the concentration of glucose is higher implies the glucose-controlled growth of Au- NPs. On modified glass slides β-D(+) glucose is detected with a sensitivity limit that corresponds to 2 x 10^"6 M.

B. Catalyzed Growth of Gold Nanoparticles in the Presence of NAD(P)H Cofactors

The method and device of the invention are further exemplified through the optical quantitative analysis of the NAD(P)H cofactors and the analysis of NAD(P)⁺-dependent biocatalyzed reactions on surfaces. A solution consisting of: (i) citrate-stabilized Au-NP (13 run ± 1.2 nm), at a concentration of 4.0 x 10^"10 M, (in particles)); (ii) HAuCl₄, at a concentration of 1.8 x 10^"4 M, and CTAB surfactant, at a concentration of 7.4 x 10^"2 M was used as seeding solution for the growth of the particles.

Figure HA shows the spectral changes of the solution upon interaction with different concentrations of NADH. Without added NADH, the solution exhibits an absorbance band at λ = 392 nm, Figure 1 IA, scale (a), characteristic to the AuCl₄ ^" component. Upon the addition of NADH, this band disappears instantaneously, and the characteristic orange color of the system is depleted. In the resulting colorless solution, the slow build-up of the particle plasmon absorbance is observed, Figure 1 IA, scales (c) - (h). As the concentration of NADH increases, the absorbance of the Au-particles increases and is shifted to longer wavelengths (from 523 nm to 530 nm). Figure HA, inset, shows the derived calibration scale depicting the absorbance changes at λ = 524 nm as the concentration of NADH increases. Figure HB shows the absorbance changes of the Au-NP seeding solution, 4 x 10^'10 M, that includes the HAuCl₄, 1.8 x 10^" ⁴ M, and CTAB, 7.4 x 10^"2 M, upon interaction with different concentrations of the NADPH cofactor. Similarly, the absorbance of the Au-particles increases and is shifted to longer wavelengths as the concentration of the reduced cofactor is elevated. Figure HB, inset, shows the derived calibration scale that corresponds to the absorbance of the Au-particles at λ = 524 nm, generated by different concentrations of NADPH. Control experiments reveal that all of the components included in the seeding solutions are essential in order to result in the enhanced growth of the Au-particles. Exclusion of the Au-NP from the

' seeding solution does not yield any gold particles upon the addition of the NAD(P)H cofactors, implying that the Au-NP seeds act as the catalysts for the growth of the particles, but the AuCl₄ ^" salt is reduced to the colorless Au(I) species by the NAD(P)H cofactors. Similarly, the CTAB surfactant is essential to stimulate the growth of the Au-NP upon the addition OfNAD(P)H.

Without being bound to theory, it is suggested that the enlargement of the Au-NP seeds by the NAD(P)H/AuCl₄ ^" solution involves two steps: In the primary step the rapid reduction of AuCl₄ ^" by NAD(P)H to the colorless Au(I)₅ eq. 1, species proceeds rapidly. In the second step, the slow catalyzed reduction of the Au(I) species by the Au-NP seeds to the Au metal particles occurs, eq. 2.

_{E χ} AuCl l +NAI⁾Ξ » Au(T) +4CI- +NAD⁺ +H⁺

_E 2 2Au(I) + NADH ^^~m > 2Au° +NAD⁺ + H⁺

The growth of the Au-NPs by the NAD(P)H cofactors was also examined on surfaces. Glass slides were functionalized with a 3- aminopropylsiloxane film, and the citrate stabilized Au-NPs, 3 nm ± 1 nm, (prepared by the reduction with NaBH₄ and further stabilized by citrate) were electrostatically bound to the surface. The resulting Au-NP-functionalized glass slides were then interacted with the growth solution that included HAuCl₄, 1.8 x 10^"4 M, CTAB, 7.4 x 10^"2 M, and different concentrations of the NADH cofactors. Figure 12 depicts the spectral changes of the glass interfaces upon interaction with different concentrations of NADH. Visually, the glass slides turn from red into dark blue colors, depending on the concentration of NADH in solution. As the concentration of NADH is elevated, the absorbance spectra of the glass surfaces reveal an increase in the absorbance band at λ = 535 nm. At NADH concentrations that are higher than 0.55 mM, the evolution of a second absorbance band at λ = 650 nm is observed. Thus, at high NADH concentrations, the growth of the Au-NP on the surface leads the contacted Au- particle aggregates giving rise to the long-wavelength absorbance band.

The glass surfaces functionalized with the Au-NPs that were interacted for the same time interval with different concentrations of NADH were further investigated using Scanning Electron Microscopy (SEM), Figure 13. From this figure it is observed that increasing concentration of NADH from 6.8 x 10^"5 M to 27 x 10^"5 M, 41 x 10^"5 M to 54 x 10^'5 M, images (A) to (D), results in the increase of the surface coverage of enlarged Au particles on the glass surfaces with particles exhibiting dimensions of 6 + 1 nm, 13 + 2 nm, 18 + 5 nm to 40 + 8 nm, respectively. At high NADH concentrations, images (E) and (F), high surface coverages of the enlarged Au-particles is observed. It is interesting to note, however, that the enlarged particles exhibit smaller dimensions, ca. 20 ± 5 nm, yet the particles touch one another forming 2D Au-particle aggregates. The latter observation is consistent with the fact that at high NADH concentrations an interparticle coupled plasmon absorbance band is observed for the aggregated nanoparticles on the surface. The SEM analysis of surface-enlarged Au-particles provides important information on the growth mechanism of the particles in the presence of the AuCl₄TNADH system: (i) At low NADH concentrations the probability of initiating the catalytic enlargement of the Au- NP seeds is low. Once Au-NP seeds are activated, they grow effectively. This is the reason that at low NADH concentrations the surface coverage of the enlarged particles is low but the particles reach dimensions corresponding to 30 ± 20 nm. (ii) At high NADH concentrations the probability of activating the growth of the Au-NP increases. This facilitates the parallel growth of numerous activated seeds, leading to smaller particles (20 + 5 nm) with a high surface coverage.

In the subsequent step, the growth of the Au-NP seeds by the NADHTHAUCI₄ ^" system was applied to analyze the substrate coupled to a NAD⁺-dependent biocatalyzed process. The fact that the growth of the Au-NP seeds proceeds in an acidic medium (pH = 4.0) rich with CTAB resulted in the compartmentalization of the analytical assay, while preventing the direct contact between the enzyme-active solutions and the Au-NP growth systems. As an example, Figures 14A and 14B depict the analysis of lactate in the presence of the NAD⁺-dependent lactate dehydrogenase, LDH, using the catalytic growth of the Au-NP seeds as a read-out method. In one compartment, the LDH-mediated reduction of NAD⁺ by different concentrations of lactate proceeds for a fixed reaction-time of 30 minutes. The resulting biocatalytic mixture that included the LDH-generated NADH was then introduced into the second compartment that included the Au-NP seeds in the growth solution. Alternatively, the biocatalytic mixture that included the enzyme-generated NADH was added to the Au-NP-aminosiloxane-functionalized glass surfaces in the presence Of AuCl₄ ^', Figure 14B. The spectral changes of the Au-NP seeds solution that included AuCl₄ ^" upon addition of the LDH-generated NADH formed in the presence of different concentrations of lactate are shown in Figure 14A. At low lactate concentrations, only the instantaneous decrease of the AuCl₄ ^" band at λ = 392 nm was observed, scales (a) - (e). At higher lactate concentrations the AuCl₄ ^" absorbance disappears instantaneously, and upon standing the slow enlargement of the NP seeds proceeds with the concomitant formation of the plasmon-band of the particles in the visible spectral region, Figure 14 A, scales (e) to (k). Figure 14 A, inset, shows the calibration scale that corresponds to the absorbance changes at λ = 524 nm, upon analyzing different concentrations of lactate. Figure 14B, scales (a) to (h), shows the plasmon bands corresponding to the enlarged particles on the surface that were formed after 60 minutes by the biocatalytically-generated NADH. As the concentration of lactate increases, the plasmon absorbance band is enhanced and shifted to longer wavelengths. Thus, as the lactate concentration is higher, more Au-NP seeds are activated towards the enlargement process, and the resulting particles reveal larger dimensions.

In a different experiment, Ag nanoparticles were used instead of gold nanoparticles. Figure 15, curve a, corresponds to the absorbance of silver particles seeds in the reaction medium (3 x 10^"10 M 15 nm Ag-NPs, 2 x 10^"4 M HAuCl₄, 2 x 10^"3 M CTAC in 0.01 M phosphate buffer) whereas curve b corresponds to the absorbance of the same Ag-NP seeds upon enlargement by the growth of a gold shell. By an addition of H₂O₂ the gold salt is reduced into crystalline gold on the Ag-NP seeds. Control experiments show that Au crytallites are not produced in absence of Ag nanoparticles. This experiment shows that the Ag seeds can be used instead of Au seeds in the H₂O₂ mediated Au crystalline growth process.

C. Catalyzed Growth of Gold Nanoparticles in the Presence of Osmium Complex

In another experiment, the results of which are showed in Figure 16, growth solutions consisted Of HAuCl₄, 1.5 x 10-3 M, chloro-Os(III) bis-2,2'- bipyridine-mono-4-pyridine carboxylic acid dichloride, 1.0 x 10-4 M, citrate, 7.5 x 10-3 M, glucose oxidase (GOx), 1.7 x 10-5 g/mL and different concentration of glucose. For the catalytic growth of the Au-NPs, the Au-NPs (13 ± 2 nm), 4.0 x 10-11 M, were added to the growth solution and the resulting absorbance spectra were recorded after 3 minutes of reaction at 30 °C. D. In situ generation of Au nanoparticle seeds 1) NADH-citrate-H₂O₂ system:

Growth and enhancement solutions consisted of HAuCl₄, 7.9 x 10-4 M, H₂O₂, 0.027 M and different concentration of NADH. For the catalytic generation and growth of the Au-NPs, citrate, 7.9 x 10-3 M, were added to the growth and enhancement solution and the resulting absorbance spectra were recorded after 5 minutes of reaction at RT. Figure 17 shows the spectral changes of the generated Au-NP produced in the solution that includes HAuCl₄ and citrate, upon the addition of different concentrations of NADH.

2) NAD⁺ dependent enzyme (glucose denydrogenase)-citrate-H₂0₂ system:

Growth and enhancement of solutions consisted Of HAuCl₄, 7.9 x 10^"4 M, H₂O₂, 0.027 M, a 100 μl of Tris buffer solution, 50 niM, pH = 9.0, that included NAD⁺, 1 x 10^"3 M, glucose dehydrogenase, 0.3 mgmL^'1 and different concentrations of glucose. For the catalytic generation and growth of the Au- NPs, citrate, 7.9 x 10^"3 M, were added to the growth and enhancement of solution and the resulting absorbance spectra were recorded after 10 minutes of reaction at 30 "C. Figure 18 shows the spectral changes of the generated Au-NP produced in the solution that includes HAuCl₄, H₂O₂, 0.027 M, citrate, NAD⁺, and glucose dehydrogenase, upon the addition of different concentrations of glucose.

Assay for the detection of tyrosinase activity

The generation of Au-NPs by neurotransmitters (e.g. dopamine, L-

DOPA), adrenaline and noradrenaline) was used to assay the activity of the

enzyme tyrosinase that catalyzes the O₂-induced hydroxylation of tyrosine to

L-DOPA. The latter product stimulates the formation of the Au-NPs. The

study has demonstrated the sensitive detection of tyrosinase activity and the enzyme could be detected with a sensitivity limit of 10 units. Since tyrosinase

is specifically expressed by melanocytes and melanoma cells and is viewed as a

specific marker for these cells, this analytical protocol is important for clinical

diagnostics. The results clearly demonstrate that the absorbance of the Au-NPs

may be an optical detection for the quantity of tyrosinase.

The tyrosinase activity as showed in Figure 19, was monitored by

recording the absorbance spectra of the Au-NPs formed in the presence of

different concentrations of tyrosinase and a fixed concentration of tyrosine. As

the concentration of the enzyme increases the absorbance of the Au NPs is

higher and blue-shifted, consistent with the fact that larger particles and small

Au-nanoclusters are formed as the product mixture. Figure 19 inset shows the

derived calibration curve. The tyrosinase is detected with a sensitivity limit of

ca. 10 units. This translates to ca. 100 μg ml^"1 of the enzyme in the sample.

Assay for the detection of nerve gases

As the concentrations of the enzyme (or substrate) increase the

hydrolysis of thioacetylcholine to thiocholine is enhanced. The latter product

acts as the reducing agent for the reduction of AuCl^4" and the deposition of gold

on the Au NP seeds. As the amount of reductant increases, larger Au particles

with increased nanoclustering on the particles surface are formed giving rise to

intensified absorbance bands (see the scheme in Fig. 20). Nonetheless, as the

concentrations of the enzyme (or substrate) increase the content of the small Au

NPs (8-30 nm) is substantially higher, and these small NPs lead to the blue

shifted, yet broader intensified, plasmon bands. These small NPs may be generated by the detachment of the thin nanoclusters associated with the

surface of the large particles.

The inhibition of the activity of AChE by following the inhibited growth

of Au NPs was monitored by using l,5-bis(4-

allydimethylammoniumphenyl)pentane-3-one dibromide or diethyl p-

nitrophenyl phosphate (paraoxon-ethyl), common AChE inhibitors that mimics

the functions of nerve gases. From absorbance spectra analysis it is concluded

that as the concentration of the inhibitor increases the enlargement of the Au

NPs is blocked as reflected by the lower intensities of the absorbance bands,

due to the lower yield of the hydrolysis product.

A surface-immobilized sensor: glass plates were functionalized with an

aminopropylsiloxane film and the Au NP seeds were bound to the surface.

Figure 21 shows the absorbance spectra of the Au NPs deposited on the

surface in the presence of AChE, 0.13 units/mL, and acetylthiocholine,

1.4xlO^'4 M, in the absence of the inhibitor, curve (a), and in the presence of

different concentrations of the inhibitor (l,5-bis(4-

allydimethylammoniumphenyl)pentane-3-one dibromide), curves (b) to (e). In

the absence of the inhibitor the surface exhibits a blue color with a maximum

absorbance at λ = 570 nm. As the concentrations of the inhibitor increase the

plasmon absorbance bands of the NPs decrease in their intensities and are blue

shifted. Thus, a surface engineered sensor that includes an active sensing

domain and a control domain may provide an effective optical strip for the

detection of nerve gases. Experimental Section for the experiments on catalytic growth of Au-NPs using AuCl₄ ^" and H₂O₂

Au-NPs (12 ± 1 run) stabilized with citrate were prepared according to the literature [15]. The concentration of the Au-NPs solutions was determined by two independent methods: (i) By the application of the particle dimensions determined by TEM and knowing the total numbers of gold ions that generate the NPs (with the assumption that all ions are reduced), (ii) By knowing the particles size and knowing the appropriate extinction coefficient. Both methods gave similar values. The Au-NPs solution was stable for at least 1 month after preparation.

Growth solutions consisted of 2 x 10^"4 M HAuCl₄ in 0.01 M phosphate buffer, pH = 7.2, 2 x 10^"3 M cetyltrimethylamonium chloride (CTAC) and either different concentrations of H₂O₂ or β-D(+) glucose with 47 μg.ml^'1 glucose oxidase (GOx). For the catalytic growth of the Au-NPs in solution, 3 x 10^"10 M Au-NP seeds (12 ± 1 nm) were added to the growth solution. The experiments were performed at ambient temperature (22 ± 2⁰C), unless stated otherwise stated. Glass slides were functionalized with 3- aminopropyltriethoxysilane as described previously and modified accordingly with the citrate-stabilized Au-NPs (12 ± 1 nm) [16]. The gold nanoparticles modified glass slide was then soaked in the above-described growth solution. The absorbance features of the resulting modified slides were recorded in water.

Absorbance measurements were performed using a spectrophotometer UV-2401PC (Shimadzu Corporation).

Au-NPs were investigated with a Transmission Electron Microscope Tecnai F20 G² (FEI Company) at 200 kV. The samples were prepared by putting a drop of the solution on a carbon coated copper grid (300 mesh) and subsequent air drying. For microscopic investigations of the modified slides one-side conductive ITO glass and a High Resolution Scanning Microscope Sirion (FEI Company) at 3 kV were used.

Experimental Section for the experiments on catalyzed growth of gold nanoparticles in the presence of NAD(P)H cofactors

Au-NPs (13 ± 2 run) stabilized with citrate and Au-NPs (3.5 ± 0.5 nm) prepared with NaBH₄ and stabilized with citrate were prepared by literature procedures [15]. The concentration of the Au-NPs (13 ± 2 nm) was determined by following the absorbance spectra, λ = 519 nm, and by using the appropriate extinction coefficient. The Au-NPs solution was used within 2-5 hours after preparation.

Growth solutions consisted of cetyltrimethylamonium bromide, CTAB, 7.4 x 10^~2 M, HauCl₄, 1.8 x 10^~4 and different concentration Of NAD(P)H. For the catalytic growth of the Au-NPs, the Au-NPs (13 ± 2 nm), 1 x 10^"10 M, were added to the growth solution and the resulting absorbance spectra were recorded after 30 minutes of reaction at 30 ⁰C.

Glass slides were functionalized with 3-aminopropyl triethoxysilane as described previously, and modified accordingly with the citrate-stabilized Au- NPs (3.5 ± 0.5 nm) [16]. The gold nanoparticles modified glass slide was then soaked in the growth solutions for lhour, 30 ⁰C, that included CTAB, 7.4 x 10^" ² M, HAuCl₄, 1.8 x 10^'4 M, different concentration of NAD(P)H. The absorbance features of the resulting modified slides were recorded in water.

The LDH mediated oxidation of lactate in the presence of NAD⁺ and using the enlargement of the Au-NPs as optical label was performed in two steps: (i) In the first step, a Tris buffer solution, 50 mM, pH = 9.0, that included NAD⁺, 1 x 10^"3 M, LDH, 0.2 mg iriL^'1 and different concentrations of lactate was allowed to react for 30 minutes, 30⁰C. (ii) In the second step 200 μl of the mixture prepared in (i) were added to 2.5 mL growth solution that included CTAB, 7.4 x 10^'2 M, HAuCl₄, 1.8 x 10^"4 M, pH = 1.8. Gold NPs (13 ± 2 nm), 1.4 x 10^"10 M, or the Au-NP functionalized slides were added as seeds to the growth solution. The absorbance spectra of the solutions or of the Au-NP- functionalized slides, were then recorded after 30 minutes of reaction at 30°C.

Experimental Section for the experiments on detection of tyrosinase activity

Materials. Dopamine, L-DOPA, Adrenaline, Noradrenaline, Cetyltrimethylamonium chloride (CTAC, 25% in water), hydrogen tetrachloroaurate (HAuCl₄₅SH₂O, 99.9%), L-Tyrosine were from commercial source (Aldrich and Sigma) and were used without futher purification. Tyrosinase (TR, from mushroom, 1.14.18.1, Fluka) was stored at -18⁰C. Ultrapure water from NANOpure Diamond™ Barastead source was used in all experiments.

Experimental conditions. Growth solutions consisted Of HAuCl₄, 2 x 10^"4 M, CTAC, 2 x 10^"3 M in 0.01 M phosphate buffer, pH = 7.2, and either different concentrations of the mentioned neurotransmitters or different concentrations of Tyrosine and Tyrosinase. All solutions were used within one hour after preparation and the experiments were performed at ambient temperature (25 ± 2⁰C).

Instrumentation. Absorbance measurements were performed using a spectrophotometer UV-2401PC (Shimadzu). Au-NPs were characterized by transmission electron microscopy, TEM, Tecnai F20 G2 (FEI) at 200 kV. The samples were prepared by placing a drop of the repective solution on a carbon coated copper grid (300 mesh) and subsequent air drying.

Experimental Section for the experiments on nerve gas sensing

Materials. Acetylcholine esterase and other chemicals were obtained from Sigma-Aldrich and were used as supplied. Au NP seeds (2 -3 run) stabilized with citrate were prepared according to the literature. The concentration of the Au NPs was determined by following the absorbance spectra, λ= 510 run, and by using the extinction coefficient 6.5xlO⁵ Cm^-1M^"1.

Acetylcholine esterase assay. 80 μL of 1.6 mM M thioacetylcholine chloride in 0.1 M Tris buffer (pH 8.0) and 4 μL of acetylcholine esterase solution in 0.1 M Tris buffer were incubated at 35 oC for 15 min. Next, 800 μL of 1.25 mM HAuCl₄ and 30 μL of 2-3 run aqueous gold NP seeds were added to give the final concentrations of thioacetylcholine chloride, 1.4xlO^"4 M, HAuCl₄ 1.1x10^" ³ M, gold NP seeds, 3.6xlO^"8 M, and after 5 min the absorbance spectrum of the resulting solution was measured.

Determination of the inhibition constant of l,5-bis(4-allyldimethylammonium- phenyl)pentan-3-one dibromide (3). 80 μL that included variable concentrations of thioacetylcholine chloride in 0.1 M Tris buffer (pH 8.0) that included variable concentrations of l,5-bis(4-allyldimethylammonium- phenyl)pentan-3-one dibromide and 4 μL of acetylcholine esterase solution in 0.1 M Tris buffer (0.13 units/mL) were incubated at 35⁰C for 15 min. Afterwards, 800 μ L of 1.25 mM HAuCl₄ and 30 μL of l.lxlO^"6 M of 2-3 nm aqueous gold NP seeds were added to the enzyme mixture. The absorbance spectra of the formed Au-NPs were recorded after 5 minutes. Inhibition of Au-NP growth on glass slides by l,5-bis(4- allyldimethylammonium-phenyl)pentan-3-one dibromide. 45 μL of 1.6 mM M thioacetylcholine chloride in 0.1 M Tris buffer (pH 8.0) containing variable concentrations of thiocholine and 16 μL of acetylcholine esterase solution in 0.1 M Tris buffer (0.13 units/mL) were incubated at 35⁰C for 15 min. Afterwards, the enzyme solution was mixed with 3.2 mL of an aqueous solution Of HAuCl₄, 1.25 mM. The Au NPs-functionalized glass slides were immersed in the resulting solution for 5 minutes. The slides were then washed with water and their absorbance was followed in water.

Claims

CLAIMS:

1. A method for the detection of the presence of an analyte in a sample, the analyte being one member of an enzyme/substrate pair, the method comprising:

(a) providing nanoparticles of a transition metal; the other member of the pair; an electron acceptor, and a transition metal salt capable to undergo a redox reaction with an electron donor thereby depositing metal on said nanoparticles;

2. A method for the determination of amount of an analyte in a sample, the analyte being one member of an enzyme/substrate pair, the method comprising:

(c) determining change in size of the nanoparticles;

(d) comparing the change in size of the nanoparticles with a calibration scale showing the relation between change in nanoparticles size to the amount of the one member under said predetermined conditions; thereby determining the amount of said one member in the sample.

3. The method of claim 1 or 2 wherein said enzyme/substrate pair is selected from oxidoreductase/oxidoreductase substrate, hydrolase/hydrolase substrate and hydroxylase/hydroxylase substrate.

4. The method of claim 3 wherein said enzyme/substrate pair is an oxidoreductase/oxidoreductase substrate pair.

5. The method of claim 3 or 4 wherein said electron acceptor is selected from O₂, Os(III) complex and NAD(P)⁺.

6. The method of claim 3 or 4 wherein said electron donor is selected from H₂O₂, Os(II) complex, NAD(P)H, transition metal complexes

7. The method of claim 6 wherein the electron acceptor is NAD⁴VNADP⁺ and the nanoparticles are formed in situ.

8. The method according to claim 6 wherein the in situ formation of the nanoparticles is by provision of conditions comprising seeds of a transition metal salt, at least one electron donor, and basic conditions.

9. The method of claim 8 wherein the at least one electron donor comprises citrate and H₂O₂.

10. The method according to claim 8 wherein the at least one electron donor comprises CTAB and ascorbic acid.

11. The method according to claim 8 wherein the basic conditions are a pH of about 10.

12. The method of anyone of the preceding claims wherein the transition metal salt is selected from Au, Ag, Cu and Pt.

13. The method of anyone of the preceding claims wherein the transition metal is gold.

14. The method of anyone of the preceding claims wherein the nanoparticles of the transition metal and the transition metal salt have the same transition metal.

15. The method of anyone of the preceding claims wherein the nanoparticles comprise gold and the salt of the transition metal is a AuCl₄ ^" salt.

16. The method of anyone of the preceding claims wherein the one member is an oxidoreductase enzyme.

17. The method of claim 16 wherein the oxidoreductase is selected from glucose oxidase, alcohol oxidase, lactate oxidase, choline oxidase, alcohol oxidase, tyrosinase and cholesterol oxidase.

18. The method of anyone of claims 1 to 15 wherein the one member is a substrate of an oxidoreductase enzyme.

19. The method of claim 18 wherein the substrate is selected from: glucose, lactate, choline, cholesterol, alcohol, tyrosin and acethyl-choline esterase inhibitors.

20. The method of anyone of the preceding claims, wherein the detection or determination of size of the nano particles is carried out optically.

21. The method of claim 20 wherein the optical detection or determination of size of the nano-particle is by determination of a change in wavelength of an electromagnetic radiation.

22. The method of claim 20 wherein the optical detection or determination of the size of the nano-particle is by determination of change of absorbance of an electromagnetic radiation.

23. The method of anyone of the preceding claims wherein the nanoparticles are immobilized on a solid substrate.

24. The method according to claim 23 wherein the solid substrate is made of a material selected from glass, conductive glass, polymers, ceramics, metal oxides and metals.

25. The method according to claim 23 wherein the immobilization is by electrostatic or covalent interactions.

26. The method according to claim 23 wherein the nanoparticles are immobilized on an electrode, a capacitor, a resistor or a non-conductive substrate present between two electrodes or wires and the detection or determination of the size or generation of the nanoparticles is carried out electrochemically or electrically.

27. The method according to claim 23 wherein the nanoparticles are immobilized on a solid support, and the detection or determination of the size or generation of the nanoparticles is carried out by measuring piezoelectric effect consisting of a change of resonance frequency of said solid support.

28. The method according to anyone of the preceding claims wherein the sample is selected from: saliva, blood, plasma, sperm, urine, cerebro-spinal fluid, sewage water, fluid obtained from industrial reactions in the food, cosmetic or pharmaceutical industry, and liquid solutions used for laboratory and research purposes, juice, wine, fruit, milk, air, breath of an individual, gas suspected of containing particles used in biological or chemical warfare.

29. The method according to claim 4 wherein the electron acceptor is NAD⁺ or NADP⁺, wherein said conditions comprise a first set of conditions enabling enzymatic reaction between NAD⁺/NADP⁺ and oxidoreductase substrate; and a second set of conditions enabling redox reaction between NADH/NADPH and transition metal salt.

30. The method according to claim 29 wherein said first set of conditions comprises a pH of about 7 to about 9 and said second set of conditions comprises a pH of about 5 or lower.

31. A kit for the detection of the presence or the amount of an analyte in a sample, the analyte being one member of an enzyme/substrate pair comprising:

(a) nanoparticles of a transition metal; the other member of the pair; a transition metal salt capable of undergoing a redox reaction with an electron donor thereby depositing metal on said nanoparticles and optionally an electron acceptor; and

32. The kit according to claim 31 wherein said enzyme/substrate pair is selected from oxidoreductase/oxidoreductase substrate, hydrolase/hydrolase substrate and hydroxylase/hydroxylase substrate.

33. The kit according to claim 31 wherein said enzyme/substrate pair is an oxidoreductase/oxidoreductase substrate pair.

34. A device for the detection of the presence or amount of an analyte in a sample, the analyte being one member of an enzyme/substrate pair, the device comprising: a solid surface having immobilized thereon in a distinct location nanoparticles of a transitional metal and optionally, also the other member of the pair and optionally also electron acceptor.

35. The device of claim 34 wherein said enzyme/substrate pair is selected from oxidoreductase/oxidoreductase substrate, hydrolase/hydrolase substrate and hydroxylase/hydroxylase substrate.

36. The device of claim 34 wherein said enzyme/substrate pair is an oxidoreductase/oxidoreductase substrate pair.

37. The device according to claim 34 wherein the solid surface is made of a material selected from glass, conductive glass, polymers, ceramics, metal oxides and metals.

38. A kit for the detection of the presence or amount of an analyte in a sample, the analyte being one member of an enzyme/substrate pair, comprising:

(a) the device of claim 34,

(b) a transition metal salt,

(c) the other member of the pair

(d) electron acceptor;

(e) optionally a calibration scale showing the relation between an optical parameter and the amount of the one member.

39. A device according to claim 34 wherein the solid surface is an electrode, a capacitor, a resistor or a non-conductive substrate between two electrodes or wires.

40. A kit for the detection of the presence or amount of an analyte in a sample, the analyte being a member of an enzyme/substrate pair comprising;

(a) the device of claim 39,

(b) a transition metal salt

(c) the other member of the pair; (d) electron acceptor;

(e) optionally calibration scale showing the relation between an electrochemical, or electrical parameter and the amount of the one member.

41. A device according to claim 34 wherein the solid surface is a piezoelectric crystal.

42. A kit for the detection of the presence or amount of an analyte in a sample, the analyte being a member of an enzyme/substrate pair comprising;

(a) the device of claim 41 ,

(b) a transition metal salt;

(c) the other member of the pair;

(d) electron acceptor;

(e) optionally calibration scale showing the relation between a microgravity parameter and the amount of the one member.