WO2014027964A1

WO2014027964A1 - Universal biosensing and bioassay devices

Info

Publication number: WO2014027964A1
Application number: PCT/SG2013/000347
Authority: WO
Inventors: Vladislav PAPPER; Terry W. J STEELE; Robert S MARKS
Original assignee: Nanyang Technological University; Ben-Gurion University Of The Negev Research And Development Authority
Priority date: 2012-08-15
Filing date: 2013-08-14
Publication date: 2014-02-20

Abstract

A photochrome-aptamer switch assay (PHASA) and biosensing device, based on it, that allows detection and quantification of analytes within minutes and removes laborious purification and washing protocols that limit some bioassays to several hours of incubation. The PHASA device comprises a single synthetic aptamer for sensing a single analyte of interest or alternatively, a series of different aptamers for sensing different preselected analytes. The aptamer is immobilised onto the surface of the device for the rapid assay of the analyte. The switchable reporter element of the PHASA device is any sensitive photochrome or fluorochrome probe, such as stilbene or diazo derivative chemically modified with the "pseudo- hapten" moiety connected to one end of the molecule. The pseudo-hapten mimics an active part of the preselected ligand or analyte, which creates strong interactions with the active site of the aptamer. The PHASA device does not rely on fluorescence intensity quantification or on fluorescence quenching. Rather, a unique feature of the switchable reporter molecule is their reporting power via an instant conformational change upon irradiation with the excitation light. This makes it unique in the sense that most fluorescent labels or probes either do not possess this intramolecular switchable nature or require the separation of adjacent fluorophores.

Description

UNIVERSAL BIOSENSING AND BIOASSAY DEVICES

Field of the Invention

[0001] The present invention relates to diagnostic testing in general, and to biosensing and bioassay platforms based on photochrome-aptamer interactions, in particular.

Background of the Invention

[0002] Increased preoccupation in diagnostic testing paved the way for the elaboration of alternative, state-of-the-art analytical devices, known as bioassay or biosensing devices or biosensors. These usually couple an immobilised biospecific recognition entity to the surface of a transducer, which converts or transforms a molecular recognition event into a measurable electrical signal, pinpointing the presence of the target analyte. Thus, the typical biosensing device comprises the following elements:

1. The "biospecific recognition entity", such as proteins, nucleic acids, viruses, cells, microorganisms, small organic molecules etc;

2. The "molecular sensing" or "reporter entity", which can be any chemical or biochemical element such as small organic molecules, peptides, proteins, nucleic acids etc;

3. The "transducer entity", which may be any physicochemical entity capable of transducing optical, piezoelectric, electrical or other signal; this physicochemical entity receives the signal from the molecular sensing entity (resulting from the interaction - of the target analyte with the biospecific recognition entity) and converts it into another signal that can be more easily measured and quantified; 4. The associated electronics or signal processors that are primarily responsible for the display and recording of the results and communication with the biosensing device.

[0003] So far, stilbene compounds have never been used as the molecular sensing entity in the bioassay or biosensing devices, although they possess all the desired features of the ideal reporter molecules, for instance chemical and biological stability, low toxicity, synthetic availability, high photochemical sensitivity, rapid response and easy regeneration.

[0004] By stilbene compounds we mean systems that are made up of stilbene units. In all compounds of this type, 1,2-ethenediyl groups link benzene rings. There are two possible stilbene isomers:

frans-stilbene (E-isomer) cz^'s-stilbene (Z-isomer)

[0005] The irans-form of stilbene is planar in both the ground and excited state. This feature is associated with a strong absorption in the UV-VIS spectrum corresponding to the excitation of n-electrons of the conjugated ethenediyl group into n*-orbitals. The introduction of substituents can result in additional weaker absorption bands close to the strong n-n* transition, which often completely or partially obscures them. The geometry of the ris-isomer is in both the ground and excited state is essentially non-planar because of the steric hindrance between two phenyl rings and the consequent large amount of the phenyl torsion that results in a blue shift of the corresponding absorption spectra.

[0006] As one of the simplest means of converting light into mechanical motion on the angstrom scale, photo-induced trans-cis isomerisation about double bonds has long been a subject of intense research. Helmut Corner and Hans J. Kuhn (1995), Vladislav Papper et al. (1997 and 2000), Waldeck (1991), and Vladislav Papper and Gertz I. Likhtenshtein (2001) discussed the mechanisms of the photoisomerisation of stilbenes and their derivatives. The conventional picture of this process has been simplified and established as a two-state, one-dimensional model, stressing torsional motion as the primary nuclear coordinate.

[0007] The trans-cis photoisomerisation of stilbenes appears quite simple; however, the detailed picture is complicated by the presence of multiple electronic states and multiple degrees of freedom. In the simple view, upon electronic excitation, the bond order of the ethylene moiety is reduced and twisting about this bond is possible during the excited-state lifetime. The multiplicity (singlet or triplet) of the excited surface, upon which photoisomerisation occurs, is not always clear. In particular, it varies with the nature of substituents on the phenyl rings and medium surrounding the stilbene molecule.

[0008] As an introductory part of the invention, reference is initially made to Fig. 1 showing a reversible light-induced trans-cis photoisomerisation of stilbenes proceeding either from the lowest exited singlet state ^at* to the twisted singlet intermediate ^ap* ("phantom") state, where there is an avoided crossing with the ground state or, alternatively, by the intersystem crossing pathway (ISC) to the biradicaloid twisted triplet state ³p* (perpendicular with respect to the C=C double bond), which nearly crosses with the ground singlet surface. It should be noted that Fig. 1 relates to prior art knowledge, and as such it merely constitutes a reference for better understanding of the present invention. Medium Effects in Photochemistry of Stilbenes

[0010] One of the most striking features of stilbene photochemistry is its essentially strong dependence on medium viscosity and temperature. Natalie Strashnikova et al. (1999) and Parkhomyuk-Ben Arye et al. (2002) provided clear evidence that the competition between fluorescence and tr ns-cis isomerisation is very sensitive to medium viscosity. Y.-P. Sun et al. (1991) supported the medium- enhanced barrier model for the photoisomerisation dynamics of substituted stilbenes in n-alkane solvents. N. S. Park and D. H. Waldeck (1994), and K. J. Smit and K. P. Ghiggino (1985) experimentally showed that the photoisomerisation dynamics of the substituted stilbenes is strongly affected by solvent polarity. W. Rettig and W. Majenz (1988) discussed different photoreaction channels in stilbene derivatives.

[0011] W. F. Mooney III (1984) et al. described photochemical reactions of surfactant frans-stilbenes in supported multilayers and films at the air-water interface. In addition, P. E. Brown (1985) et al. researched photoisomerisation and fluorescence of surfactant and hydrophobic stilbenes in homogeneous solution. B. Suddaby (1985) et al. used surfactant and hydrophobic derivatives of trans- stilbenes as probes of vesicle and micelle solubilisation sites. Arti Ahluwalia et al. (2002) used specific stilbene-antigen derivatives for determination of orientation of solid phase antibodies.

[0012] Gertz I. Likhtenshtein et al. (1992 and 1996) and Vladislav Papper (2000) developed the fluorescence-photochrome method for investigation of local medium effects and phase transitions in biological membranes and on solid surfaces. The method is based on monitoring the trans-cis photoisomerisation kinetics of stilbene derivative incorporated into object of interest. They showed that the apparent rate constant of the trans-cis photoisomerisation in viscous media, for instance, in biological membranes, is controlled by the medium relaxation rate.

[0013] Natalie Strashnikova et al. (1999) and Vladislav Papper (2000) applied the above method to investigation of quartz solid surfaces modified with the stilbene molecules. Investigation of microviscosity effect on the photoisomerisation of irans-4,4'-bis-brommethylstilbene immobilized onto quartz plates coated with lysozyme was carried out by changing the relative concentration of glycerine in a glycerine-water mixture used as surroundings. The apparent photoisomerisation rate constant of the process was calculated using steady-state approximations, and found to be 3-4 times less for the immobilised stilbene molecule than for the same molecule in a free state in solution. That indicated that the surface and protein itself sterically hinder the rotation of the stilbene fragment around the olephinic double bond in the excited state.

Fluorochrome Immunoassay

[0014] Molecular recognition elements are organic molecules that bind targets or haptens with high affinity and specificity. A number of methods are employed, but by far the most popular and utilized are antibodies.

[0015] Oren Chen et al. (2003) investigated molecular dynamics of an antibody binding site by using the aforementioned fluorescence-photochrome method. Following the above investigation, Oren Chen et al. (2008) developed fluorochrome immunoassay (FCIA) based on the conjugation of a particular trans- stilbene derivative to an antibody of interest. This irans-stilbene derivative is a fluorescent_;photochrome dye and labelled analogue to the native antigen of the particular antibody. [0016] Theoretical considerations and existing experimental data indicate that the frans-stilbene-hapten complex suffers serious steric hindrances and constraints within the antibody binding site and its environment. The stilbene molecule being squeezed cannot undergo any geometrical changes, which normally occur upon high-frequency light irradiation at its excitation maximum. As a result, the highly fluorescent irans-stilbene molecule cannot isomerise into its non-fluorescent cis- form. Consequently, the fluorescence decay rate (photoisomerisation rate) becomes very low compared to that of the unbound irans-stilbene molecule measured in solution. Using the above method, Oren Chen et al. (2008) demonstrated how to prevent photoisomerisation of the stilbene compound when it is bound to a specific antibody and to utilize this phenomenon for the development of a rapid and potentially sensitive immunoassay in solution.

[0017] FCIA has a number of essential advantages over the currently and widely used ELISA and other assays, such as FRAT, LILA-FRAT, FPIA, FRTIA and LILA-FIA. Being a non-separation and rapid immunoassay producing results within a few minutes, it is essentially based on time-dependent fluorescence emission in-situ. FCIA made it possible to improve the specificity and sensitivity of the aforementioned assays for extremely low concentrations of the tested material up to a ppb range in air, water wastes and from hands. FCIA does not need polarization equipment and is not markedly influenced by light scattering effects. It is applicable to antigens of higher molecular weight and does not require preliminary labeling of both antigen and antibody. FCIA is not limited by a molecular distance critical for the resonance energy transfer, and can be further expanded for analyses of enzymes and receptors including adaptation to fibre- optic techniques. It is very simple, fast and non-separation immunoassay, requiring minimal training to perform and using automatic data tracking. Eventually, FCIA is able not only to identify the analyte, but also to measure its concentration in a sample by a competitive binding of the analyte present in the sample to the active site of the antibodies occupied by the frans-snTbene-hapten molecules.

[0018] In spite of all its aforementioned advantages, FCIA is not commercially available and applicable. Pursuing antibodies against different analytes, including endocrine disrupting compounds, such as Bisphenol A, toxins, viruses, explosives,, drugs etc., is an issue. Antibodies against specific analytes of interest may not be instantly and constantly available (e.g. monoclonals). The production of antibodies uses biological systems. To produce antibodies, the induction of an immune response is necessary. However, this procedure might discriminate target proteins that has similar structure to endogenous protein or toxic compounds that would kill the animal. Another complication for in-vivo production of antibodies is that the antibodies can only work under physiological conditions. This restricts the range of application and function of antibodies.

[0019] It is worthwhile to mention here that the last, but not the least problem of sticking FCIA to the use of antibodies is that the same antibodies produced by different animals may significantly differ in their structure and functionality. As a result, any.bioassay or biosensing system based on FCIA for detection of a certain analyte would differ in its sensitivity, and might be difficult to calibrate and universalize.

Aptamers

[0020] While antibody recognition has been the gold standard, numerous problems mentioned above abound towards new designs, limited protein shelf lives, and manufacturing scale-up. These problems can be surmounted, but only through laborious research programs at great cost. [0021] The aforementioned limitations of antibodies can be easily overcome by replacing them with aptamers. The main advantage of aptamers is their in-vitro selection process, whereas the production of antibodies uses biological systems. By isolating aptamers in-vitro, an aptamer can be produced for any target molecule.

[0022] Aptamers are different from antibodies, yet they mimic properties of antibodies in a variety of diagnostic formats. Aptamers are essentially ligand- binding oligonucleotide chains based on single-stranded DNA (ssDNA) or RNA (ssRNA). They introduce a new class of molecular recognition elements used in therapeutic and diagnostic applications that rival antibodies in both affinity and selectivity when bind to pre-selected targets, such as small organic molecules, proteins or viruses. These nucleic acid sequences can assume a variety of shapes due to their propensity to form helices and single-stranded loops, explaining their versatility in binding to diverse targets.

[0023] The aptamers is gaining acceptance and usage in numerous biomedical fields, including sensors, therapeutics, and diagnostics. S. Song et al. (2008) and E. J. Cho et al. (2009) reviewed the application of aptamers as biosensors. Since aptamers are based on oligonucleotides such as RNA and DNA, techniques for their synthesis and characterization are mature technologies. They do not suffer from the problems of antibodies previously mentioned. Moreover, aptamers have long shelf lives, can be rapidly screened by the SELEX method for new designs, and can be rapidly scaled for manufacturing. The SELEX selection process usually takes about 8 weeks for the development of an aptamer and takes up to 6 months for antibody selection. Therefore, this will save a large amount of time compared to the in-vivo selection process for antibodies.

[0024] Another complication for in-vivo production of antibodies is that the antibodies can only work under physiological conditions. This restricts the range of application and function of antibodies. Aptamers on the other hand can be optimized for any conditions, they can also be manipulated to bind different region of the target in different conditions. Also, aptamers are more stable at high temperature and they can be regenerated easily after denaturation. The quality of aptamers is more consistent than antibodies because they are synthesized chemically and then purified.

[0025] In view of the above, aptamers bring considerable advantages over antibodies towards biosensors. They are much smaller than antibodies - generally an order of magnitude lower molecular weight, allowing higher surface densities on optical fibers, for example. Their molecular structure or 'shape' is more stable, allowing mixtures of organic and aqueous solvents for analyte testing. They are also relatively inexpensive to produce. These features make them ideal for use in the bioassay and biosensing device according to the embodiments of the invention.

Summary of the Invention

[0026] Various embodiments of the invention provide devices, systems and methods for bioassay and biosensing including rapid detection of a preselected ligand or analyte in a liquid sample such as waste water or body fluid or from solid surfaces and hands.

[0027] The present invention relates to a universal bioassay and biosensing device that allows detection and quantification of analytes within minutes and removes laborious purification and washing protocols that limit some bioassays to several hours of incubation, for example ELISA detection of analytes.

[0028] In one embodiment, the universal sensing device may be an optical, electrochemical or acoustic biosensor, microfluidic chip, magnetic particles, nanostructured surfaces, for example nanoantennas, or any other bioassay or biosensing platform. The device may be a lateral-flow strip or even a microtiter plate pre-treated with reagents for use in the commercial fluorescence microtiter plate reader. The microtitre plate may be black, white or optionally have a substantially transparent portion.

[0029] In another embodiment, the universal sensing device may comprise a single synthetic aptamer - not antibodies which are typically animal derived - for sensing a single analyte of interest or alternatively, a series of different aptamers for sensing different preselected analytes. Besides ease of manufacturing, the aptamer-based bioassay can perform in a range of environmental conditions such as aqueous or mixtures of aqueous/ organic solvents. This extends its utility into 'dirty' environments other bioassays are incapable of operating in. The aptamer may be immobilised onto the surface of the sensing device for the rapid assay of an analyte, introduced into solution taken for analysis or sprayed over the tested surface, similar to photographic developer.

[0030] In a further embodiment, the universal sensing device comprises the "switchable reporter" element. The reporter element is any sensitive photochrome or fluorochrome probe, such as stilbene or azo derivative chemically modified with the "pseudo-hapten" moiety connected to one end of the molecule. The pseudo-hapten mimics an active part of the preselected ligand or analyte, which creates strong interactions with the active site of the aptamer. It can be any small organic molecule, such as aniline and its carboxyl derivatives for serological studies, trinitrotoluene and picrates for forensic diagnostics, heroin, signal toxins, for example aflatoxin, parasitic shellfish poisoning toxins (PSP), and cocaine for drug detection and monitoring, or any polypeptide of interests. It can be also proteins, viruses and endocrine disrupting compounds, such as Bisphenol A.

[0031] In contrast to antibodies, where haptens elicit immune responses when attached to a large carrier such as a protein; the pseudo-haptens do not elicit an immune response, but serve as a diagnostic tool. Once the pseudo-hapten forms a complex with the corresponding aptamer, the sensing device is ready for its operation.

[0032] The switchable stilbene compounds have never been applied towards biosensing devices and bioassays, although they possess all the desired features of the ideal and universal reporter molecules. This includes chemical and biological stability, low toxicity, synthetic availability and versatility, high photochemical sensitivity, rapid response, and easy regeneration. The universal bioassay and biosensing device of the present invention does not rely on fluorescence intensity quantification or on fluorescence quenching. Rather, a unique feature of stilbenes is their reporting power via an instant conformational change upon irradiation with the excitation light. This makes the stilbene switches unique in the sense that most fluorescent reporters (labels or probes) either do not possess this intramolecular switchable nature or require the separation of adjacent fluorophores.

[0033] In a further embodiment, the switchable reporter element changes its spatial molecular configuration upon excitation with the UV-VIS light. As a result, the molecule undergoing geometrical changes may be "switched on" or "switched off" attaining certain isomeric form, and hence, becoming radiative or not, dependent on its internal electronic structure.

[0034] In a particular embodiment, the switchable reporter element is the fluorescent irans-stilbene derivative, which is able to adopt the non-fluorescent cis- form upon high-frequency irradiation at the excitation maximum.

[0035] In one of the embodiments, the surface of the device maybe pre-coated with metaljayer or metal particles to enhance fluorescence signal from the reporter molecule. In this case, the reporter molecule is chemically bound to or immobilised onto said metal layer or metal particles via chemical crosslinker of a suitable length capable of providing the maximum enhancement effect. The selected aptamer is then conjugated to said reporter molecule, forming a stable complex with the latter.

[0036] In another embodiment, the bioassay or biosensing device may include a time measuring element which allows using the standardized calibration time for automatic performance of measurements and automatic data tracking. The time measuring element may be any type of digital or analogue clocks for performing the diagnostic tests in a specific interval of time.

[0037] In a particular embodiment, the universal bioassay or biosensing device may include an UV-VIS detector or fluorescence detector, such as photodiode or photomultiplier tube.

[0038] In still a further embodiment, a system of the invention comprises:

■ A universal bioassay or biosensing device;

^■ An external detecting instrument, which is able to detect spectrophotometric changes (e.g., fluorescence microtitre plate reader or fibre optic UV-VIS spectrophotometer); and

^■ A computing platform or workstation able to store, process, display, or analyse the received data.

[0039] Some embodiments of the invention may include a diagnostic method based on the performed assay. The method comprises the following steps:

■ Collecting the sample from the surroundings (wastewater, body fluids, solid surfaces, skin or hands);

^■ Performing the assay using the universal bioassay or biosensing device for the predetermined period of time; and

^■ Acquiring data such as fluorescence decay curves or intensity data, of the bioassay or biosensing device. [0040] The method may further optionally include transmitting the acquired data, analysing the data, and/ or other suitable operations.

[0041] Various embodiments of the invention may allow various benefits, and may be used in conjunction with various applications. The details of one or .more embodiments are set forth in the accompanying figures and the description below. Other features, objects and advantages of the described techniques will be apparent from the description and drawings and from the claims.

Brief Description of the Drawings

[0042] The present invention will be understood and appreciated more fully from the following detailed description taken in conjunction with the appended figures. Various exemplary embodiments are well illustrated in the accompanying figures with the intent that these examples not be restrictive. It will be appreciated that for simplicity and clarity of the illustration, elements shown in the figures referenced below are schematic. Of the accompanying figures:

Fig. 1 is a schematic representation of an electronic states and molecular orbitals of the stilbene molecule involved in the light-induced trans-cis photoisomerisation.

Fig. 2: shows _^the stilbene fluorescent switch that exists in two molecular configurations: the fluorescent irans-stilbene molecule is switched to the non- fluorescent cz^'s-stilbene by UV excitation light.

Fig. 3 illustrates the PHASA quantification based on the fluorescence kinetic decay in two cases: A) the fluorescent frans-stilbene is excited with UV light, causing a molecular switch into the corresponding a^'s-isomer (the fluorescence decay first- order rate constant, k (arbitrary chosen at 30 sec), is dependent on the initial irans- stilbene concentration before irradiation and on local environment, and is independent of fluorescence intensity); B) the irans-stilbene is sterically hindered, the trans-cis isomerisation is prevented, and the fluorescent switch is 'jammed' in the 'on' position.

Fig. 4 is a schematic representation of the stilbene-aptamer assay according to Method 1, operative in accordance with an embodiment of the present invention;

Fig. 5 is a schematic representation of the stilbene-aptamer assay according to Method 2, operative in accordance with an embodiment of the present invention.

Detailed Description of the Invention

[0043] In the following description, various aspects of the invention will be described. For purposes of explanation, specific configurations and details are set forth in order to provide a thorough understanding of the invention. However, it will also be apparent to one skilled in the art that the invention may be practiced without the specific details presented herein. Furthermore, well-known features may be omitted or simplified in order not to obscure the invention.

[0044] It should be noted that although a portion of the discussion may relate to bioassay or biosensing diagnostic devices, systems, and methods, the present invention is not limited in this regard, and embodiments of the present invention may be used in conjunction with various other in-vitro or in-vivo sensing and diagnostic devices, systems, and methods. As such, some embodiments of the invention may be used, for example, in conjunction with toxicological tests for environment or for a human, in-vitro or in-vivo sensing of pH, temperature, pressure and/or electrical impedance, in-vitro or in-vivo detection of a substance or a material using different diagnostic techniques or combination thereof, in-vitro or in-vivo detection and imaging of a medical condition or a pathology, in-vitro or in- vivo acquisition or analysis of data, and/ or various other in-vitro or in-vivo sensing, diagnostic and imaging devices, systems, and methods. Some embodiments of the invention may be used not necessarily in the context of in vivo imaging or in vivo sensing.

[0045] In accordance with embodiments of the present invention, the device is based on a novel bioassay and biosensing technologies that allows limitless ligand selectivity and high sensitivity in a small portable instrument without the use of chemical reagents or consumables. Photochrome-aptamer switch assay (PHASA) is based on the conjugation of a particular switchable reporter element to any aptamer of interest. The examples of the reporter molecules are stilbene or azo derivatives, or any another photochrome probe capable of changing its molecular configuration upon irradiation at the excitation maximum. The reporter molecule acts as both a photochrome probe and a labelled analogue to the highly selective aptamer binding moiety.

[0046] The switchable reporter molecule is designed to detect and quantitate any ligand or analyte of interest. For example, the switchable reporter molecule can be 2,4,6-trinitrophenyl (TNP) derivative or Bisphenol A-valeric acid derivative of 4'-dimethylamino-4-aminosulbene (TNP-DMAAS and BPAVA-DMAAS), which are shown below, for forensic and water toxicit assessment:

TNP-DMAAS

BPAVA-DMAAS

[0047] Technology is conceived to offer higher cost-performance in the detection and identification of TNT, picric acid and similar trace explosives, as well as trace amounts of Bisphenol A in waste water.

[0048] Backbone of the novel technology relies on the photoisomerisation of a fluorescent isomer of a reporter molecule to a non-fluorescent isomer. Stilbene compounds so far have never been used as the molecular sensing entity in the biosensing devices and bioassays, although they possess all the desired features of the ideal reporter molecules, for instance chemical and biological stability, low toxicity, synthetic availability, high photochemical sensitivity, rapid response and easy regeneration.

[0049] Stilbenes are capable of changing their molecular configuration upon irradiation at the excitation maximum, as shown in Fig. 2. As an example, the highly fluorescent frans-isomer of TNP-DMAAS is photoisomerised to the corresponding non-fluorescent a^'s-isomer under irradiation at λ = 374 nm, which is excitation maximum of the frans-isomer. A unique feature of stilbenes is their reporting power via an instant conformational change upon irradiation with the excitation light. This makes the stilbene switches unique in the sense that most fluorescent reporters (labels or probes) either do not possess this intramolecular switchable nature or require the separation of adjacent fluorophores (like, for example, in FRET assay).

[0050] Design of a bioassay or biosensing device utilising PHASA is based on matching specifically to an aptamer of interest the specific reporter molecule containing the "pseudo-hapten" moiety. The reporter molecule acts as both a fluorescent-photochrome dye and a labelled analogue to the highly selective aptamer binding moiety. In case of TNP-DMAAS, the synthetic reporter molecule merges fluorescent irans-DMAAS and the pseudo-hapten moiety, 2,4,6- trinitrophenyl group (TNP). The binding event of the reporter molecule and its reporting molecular configuration within the binding site of the specific aptamer can be readily monitored by a single steady-state fluorescence technique.

[0051] The reporter molecule is unique in the way that it can reversibly adapt in solution to any of its stereochemical configurations upon UV irradiation at the excitation maximum of the corresponding isomer. In case of a stilbene molecule, the stereochemical frans-isomer is strongly fluorescent while the cz^'s-isomer is non- fluorescent. The light-induced process of tr ns-cis photoisomerisation acts like a quenching, funnel on fluorescence of the ligand-stilbene reporter molecule, which is typically equilibrated in solution toward a more stable frans-isomer. Hence, irradiation of the fluorescent frans-form of the reporter molecule at the excitation maximum produces a substantial change of the fluorescence emission that can be monitored as a time trace (kinetic decay) signal or emission gradient, which is illustrated in Fig. 3(A).

[0052] As noted above, Natalie Strashnikova et al. (1999), Vladislav Papper (2000) and Oren Chen et al. (2003) clearly demonstrated that the reaction dynamics of the trans-cis photoisomerisation reaction is strongly dependent on the properties of external environment. Theoretical considerations and existing experimental data indicate that the reporter molecule could have serious steric constraints for the light induced trans-cis isomerisation within the binding site and its environment of the properly selected aptamer. Therefore, the irans-isomer of the reporter molecule being squeezed cannot undergo any geometrical changes, which normally occur upon high-frequency light irradiation at its excitation maximum. As a result, the highly fluorescent irans-isomer cannot be transformed into its non-fluorescent cis- form, and hence the fluorescence decay (photoisomerisation) rate significantly drops compared to that of the unbound irans-isomer in solution, as schematically shown in Fig. 3(B).

[0053] In a particular embodiment, the method for detection of an analyte in a sample and measuring its concentration comprises a competitive binding of the analyte molecules present in the sample to the binding site of the specific aptamer occupied by the reporter molecule. Replacement of the fluorescent irans-isomer of the reporter molecule with the analyte in the binding site of the aptamer results in a significant acceleration of fluorescence decay of the reporter molecule upon irradiation at its excitation maximum. The latter connects to the apparent trans-cis photoisomerisation rate constant of the reporter molecule that is experimentally measured by the steady-state fluorescence technique.

[0054] Reference is now made to Fig. 4, which schematically demonstrates the photochrome-aptamer switch assay (PHASA) with stilbene-aptamer. In a specific embodiment, the PHASA is performed according to Method 1, which is described below, and where the reporter molecule is not covalently bound to the aptamer but only allowed to form a complex with the binding site of the aptamer. In this format, upon addition of the corresponding reporter molecule to its aptamer of interest, the reporter molecule forms a complex with the aptamer. Being squeezed in the aptamer binding site, the reporter molecule exhibits slow fluorescence decay upon irradiation at the excitation maximum of the irans-isomer. [0055] Introduction of a sample solution containing an analyte, results in the competitive analyte-aptamer complex formation. This replacement reaction results in release of the fluorescent reporter molecule from the aptamer active site into solution, the event that is detected as a rapid decay of fluorescence on irradiation with the excitation light. In the format of Method 1, there is a certain degree of freedom left for the reporter molecule to isomerise inside the aptamer's binding site. Nonetheless, this method eliminates the additional step of covalent binding the reporter molecule to the aptamer during preparation of the bioassay or biosensing device.

[0056] Reference is now made to Fig. 5, which shows PHASA with stilbene- aptamer according to Method 2, where the reporter molecule is covalently bound to the aptamer in the platform preparation step. In this format, after addition of the corresponding reporter molecule to its aptamer and formation of the complex with the aptamer, the reporter molecule is photoactivated to form a covalent bond with the aptamer in the vicinity of the binding site. An example of such photoactive reporter molecule is 2,4,6-trinitrophenyl (TNP) derivative of 4'-aminomethyl-4- stilbene-trifluoromethyldiazirine (TNP-AMSD):

TNP-AMSD

[0057] ^"The photoactivation of the diazirine derivative involves irradiation of the molecule at λ = 320-350 nm. As a result, the diazirine ring is split upon photolysis forming the active carbene radical, which attacks any C-H bonds in the vicinity of the molecule and forms a new covalent bond through addition reaction inside the binding site of the aptamer.

[0058] Being bound and rigidly fixed in the aptamer binding site, the reporter molecule exhibits almost no fluorescence decay upon irradiation at the excitation maximum of the frans-isomer. Introduction of a sample solution containing an analyte, results in the competitive analyte-aptamer complex formation, thereby releasing one end of the reporter molecule into solution. Consequently the reporter molecule is able to partially photoisomerise under the excitation light, the event that is detected as the fluorescence quenching and appearance of the fluorescence decay. In the format of Method 2 there is no freedom left for the reporter molecule to isomerise inside the aptamer's binding site, and the analyte molecule in the tested solution switches on the fluorescence decay. This method however requires the additional step of covalent binding the reporter molecule to the aptamer during preparation of the bioassay or biosensing device.

[0059] In a further embodiment, the method for calibration of the bioassay or biosensing device involves the steady-state fluorescence measurements of the switchable. reporter molecule in solution in a conformational free state and within a sterically hindered binding site of aptamer, and comprises the following steps:

1. Steady-state fluorescence measurements of the reporter molecule in solution and in a complex with an aptamer in order to obtain two limit (end) points of the calibration curve (the "uncomplexed" state and the "complexed" state of the reporter molecule, as shown on Figs. 4 and 5).

2. Introducing the sample analyte of different known concentrations to achieve the competitive formation of the complex aptamer-analyte monitored by steady-state fluorescence kinetics of the reporter molecule. 3. Calculation of the first-order trans-cis photoisomerisation rate constant for both the uncomplexed and complexed reporter molecule, and for different known concentration of the tested analyte.

4. Calibration of the system with the trans-cis photoisomerisation rate constant as a function of the analyte concentration at arbitrary fixed time, which is defined as a "calibration time" for field tests.

[0060] Calibration procedure described above is based on direct correlation of the photoisomerisation rate constant for the reporter molecule to the analyte concentration. It may be applied for control tests not only in sample solution but also in biological fluids and wastewater, thereby resulting in standardisation of all tested liquids in the field. The competitive binding of an analyte from the sample solution is detected as an acceleration of the fluorescence decay on irradiation with the excitation light. The exact value of the analyte concentration in the sample is obtained from the calibration of the system for the "calibration time" for all bioassay and biosensing devices of the same type (based on the same platform, such as optical biosensor or microliter plate, and for the same analyte). The calibration data for different bioassay and biosensing devices (used for detection of different analytes) may be further compiled and form a database or data library for testing different analytes using the same universal bioassay or biosensing platform of the present invention.

[0061] In another embodiment, the universal biosensing device may be an optical, electrochemical or acoustic biosensor or any other biosensing platform.

[0062] In still another embodiment, the bioassay device may be a microfluidic chip or microtiter plate pre-treated with reagents for use in the commercial fluorescence microtiter plate reader. The microtiter plate may be black, white or have a substantially transparent portion. [0063] In yet further embodiment, the method for manufacturing of the biosensing device comprises the following steps:

1. Surface modification of a transducer element or solid support of a biosensing device, such as quartz fibres of optical biosensors, electrodes of amperometric biosensors, metal surfaces of acoustic sensors, and polymeric surface of microtiter plates or lateral flow strips, by treatment with suitable activating, cross-linking and conjugating reagents;

2. Coating of a transducer element or solid support of a biosensing device with the selected aptamers.

3. Addition of the reporter molecules to coated with the aptamers surface of a transducer element or solid support of the biosensing device coated with the aptamer to form the complex of the reporter molecules with the aptamers, thereby yielding the biosensing platform.

4. Washing the prepared biosensing platform with buffers and organic solvents and drying in vacuum.

[0064] Towards the PHASA biosensor, aptamers are inherent to the initial design as too many disadvantages with antibodies are foreseen:

a) Antibodies will likely denature in organic solvents/ glycerol environments where the fluorescence quantum yield for the reporter molecules is the highest;

b) Antibodies decomposition too fast to be useful, as they have limited shelf-lives in the dry or hydrated state;

c) Any antibody (or protein having tryptophan amino residues) would have strong background fluorescence at the wavelengths needed for stilbene excitation, decreasing the signal to noise ratio, and limiting sensitivity. [0065] In addition, there are numerous biotech companies existing on the market, such as Aptagen, Aptsci and Neoven ures, that sell commercially available aptamers for over 150 targets, thereby allowing the PHASA biosensor platform to be quickly developed towards numerous industries. Even if the aptamer is not commercially available, it can be designed and manufactured in 6-8 weeks for a reasonable cost, which would be unfeasible (if not impossible) for antibodies.

[0066] Eventually, aptamers allow more novel designs and continued development of the next generation PHASA biosensors. The completely synthetic preparation of DNA/RNA aptamers can allow tailored properties towards their end applications, such as bacterial nuclease resistance for ground water biosensors based on the PHASA platform, resistance to aggressive environmental conditions (heat, acidity, organic solvents) or even immunogenicity in blood circulating devices.

[0067] The major advantage of the universal bioassay or biosensing device of the present invention over other bioassays and biosensing devices available today on the market is its almost immediate detection of an analyte. Since the photoisomerisation process of the reporter molecule is very rapid and may be standardised for 30 sec calibration time, the overall test for the presence and concentration of an analyte may require only several minutes. For comparison, the regular ELISA test for explosives in the field may take up to 6 hours.

[0068] Also, antibodies are too bulky compared to aptamers. The former have the molecular volumes 3-4 orders of magnitude larger than the binding ligand of interest, limiting high density, and thereby preventing low intensity fluorescence measurements. Due to their small size, stability in organic solvents, and high ligand-specificity, aptamers are a more logical choice for bioassays and biosensing devices than antibodies. [0069] Another advantage of the PHASA platform is based on the fact that no separation of the reagents and bound aptamers before and after measurements is required. It is essentially one-step procedure. The system is self-contained and does not need to be regenerated. The biosensor can operate not only in aqueous solutions but also in a solvent mixtures, heavily contaminated heterogeneous wastes and biological mediums collected in a single tube, without precipitation or washing steps. This biosensor permits not only the detection of an analyte in a sample but also determination of its concentration.

[0070] Also, the bioassay and biosensing device of the present invention is universal in sensing different types of different ligands (analytes) in different applications. For example, groups of microwells on the same microtiter plate can be modified with different aptamers-reporter molecules for simultaneous sensing of different analytes.

[0071] In yet a further embodiment, the biosensing device may include a time measuring element which allows using the standardised calibration time for automatic performance of measurements and automatic data tracking. The time measuring, element may be any type of digital or analogue clocks or sensors for performing the diagnostic tests in a specific interval of time.

[0072] In a particular embodiment, the universal biosensing device may include an UV-VIS detector or fluorescence detector, such as photodiode or photomultiplier tube.

[0073] In still another embodiment, a system of the invention comprises:

■ A universal bioassay or biosensing device;

■ An external detecting instrument, which is able to detect spectrophotometric changes, for example fluorescence microtiter plate reader or fibre optic spectrophotometer; and ^■ A computing platform or workstation able to store, process, display, or analyse the received data.

[0074] Some embodiments of the invention may include a diagnostic method based on PHASA. The method includes the following steps:

■ Performing the assay using the universal bioassay or biosensing device for the predetermined period of time; and

^■ Acquiring data such as fluorescence decay curves or intensity data, of the bioassay or biosensing device.

[0075] The method may further optionally include transmitting the acquired data, analysing the data, and/ or other suitable operations.

[0076] In some embodiment, the biosensing device may be fully autonomous and self-contained. For example, a device according to some embodiments may be a microfluidic chip or lab-on-chip where all the components are substantially contained within the chip, and where the device does not require wires or cables in order to receive power or transmit the signal, for example.

[0077] In another embodiment, the bioassay device may be a lateral flow strip based on PHASA. In this case, the aptamers are immobilised over the strip surface in a particular area called "the capture zone", and the reporter molecules are added to form a complex with the aptamers in this zone. The strip may be dipped into the tested analyte solution providing the competitive binding between the analyte and reporter molecules.

[0078] While most lateral flow immunoassays are only capable of providing a qualitative result, it is possible to obtain a certain degree of quantification by measuring the amount of conjugate bound to the capture zone. This may be done by using a dedicated sensor capable to measure the intensity of the fluorescent test line. By utilizing unique wavelengths of light for illumination in conjunction with either CMOS or CCD detection technology, a signal rich image can be produced of the actual test lines, which can be further served for calibration of the system as a function of the analyte concentration. Using image processing algorithms for a particular PHASA system, line intensities can then be correlated with analyte concentrations. More sophisticated techniques, such as fluorescent dye labelled conjugates, may also be implemented in order to improve the quantitative potential of the instant analytical method.

[0079] In some embodiment, a computing platform or workstation may use the WiFi or Bluetooth technology to transmit the fluorescence data for analysis. The computing platform may include, for example, a Central Processing Unit (CPU), a Digital Signal Processor (DSP), a microprocessor, a controller, a chip, a microchip, a controller, circuitry, an Integrated Circuit (IC), an Application- Specific Integrated Circuit (ASIC), or any other suitable multi-purpose or specific processor, controller, circuitry or circuit.

[0080] . According to another embodiment, a microprocessor chip may be a part of the biosensing device and may calculate or provide a quantitative determination of the fluorescence intensity changes. This information may be transmitted to the computing platform via wireless technologies. Such processing may be performed substantially in real-time or may be performed offline, e.g., using post processing operations.

[0081] Metal-enhanced fluorescence (MEF), which refers to near-field short- range (0-30 nm) fluorophore-metal interactions, achieves significant fluorophore emission signal enhancement. The exponentially decreasing electromagnetic field generated from oscillating plasmons partly dictates a distance-dependent energy coupling interaction between an excited-state dipole and induced surface plasmons. Partial energy of the excited chromophores transfers to the metallic surface plasmons, which in turn, radiate the coupled quanta, at the same wavelength as the fluorophores, but with a slight spectral distortion on the red edge of the spectra.

[0082] According to a particular embodiment, metal or metal beads are initially deposited onto the surface of said bioassay or biosensing device followed by covalent attachment of said switchable reporter molecule. The deposited metal used in the MEF method is selected from the list of silver, gold, nickel, platinum, copper, chromium or any other metal suitable for creating the fluorescence enhancement effect. For example, the deposition of silver on 96-well plates occurs by mixing a solution of sodium hydroxide and ammonium hydroxide in a continuously stirred solution of silver nitrate at room temperature followed by cooling the mixture in an ice bath for 10 min and then adding a solution of D- glucose for an additional 3 min on ice. After 20 min of heating at of 40 °C, the solution is poured into the 96-well plates and distilled water added to prevent oxidation. Optimal deposition time is determined by calculating the enhancement factor (EF) of each well relative to the control emission.

[0083] The covalent attachment of said switchable reporter molecule to a metal layer or metal particles can be done using any commercially available cross- linker of a suitable length following the standard protocol, such as EDC-mediated coupling reaction in the presence of NHS, or esterification of the carboxylic acid using 0-(N-succinimidyl)-l,l,3,3-tetramethyluronium tetrafluoroborate (TSTU). The length of the cross-linker is determined experimentally as a function of the fluorescence enhancement. In a specific embodiment, the metal is gold and the cross-linker is 11-mercaptoundecanoic acid.

[0084] In a further embodiment, the kit comprises the bioassay or biosensing device having the surface coated with a metal layer or metal particles, the switchable reporter molecule attached to said metal layer or metal particles, a single aptamer or plurality of different aptamers for sensing a single analyte or plurality of different analytes, said aptamer or aptamers are capable of forming a stable complex with said reporter molecule, and a manual for using said kit.

[0085] Aptamer immobilization affects an adaptive binding. As an example and as working model, malachite green (MG) aptamer has previously been synthesized into oligo-aptamers - consecutive repeats of dimers to decamers. The loss of 'degree of freedom' (steric hindrance) mimics surface immobilization. Interestingly, the mimicked immobilization increased binding of malachite green. Computational modelling allowed us to optimize the immobilization and to screen various frans-stilbene-ligands that may offer different binding properties than solution bound MG-aptamer.

[0086] The PHASA bioassay has many paths where it could be optimized for advanced features, such as reversible cis- to frans-stilbene generation for real-time biosensor applications. This advanced design covalently binds irans-stilbene directly to one of the 38 nucleotides in MG-aptamer. Modelling guides the optimal nucleotiderstilbene generation to have minimal effects on the normal adaptive binding.

Advantages over existing methods and devices

[0087] To sum up, the PHASA technology is based on the combination of two versatile molecules:

1) The report molecule, in particular, frans-stilbene derivative, which has chemical and biological stability, low toxicity, synthetic availability, high photochemical sensitivity, rapid response, and easy regeneration; and

2) The aptamer, which can be made in a synthetic protocol to bind virtually any hapten (small molecule alone or part of a larger entity).

1. Rapid detection in several minutes vs. ELISA several-hours protocol

[0088] The major advantage of the universal biosensing device of the present invention over other biosensing devices currently available is its almost immediate detection of an analyte. Since the photoisomerisation process of the reporter molecule is very rapid and may be standardised for 30 sec calibration time, the overall test for the presence and concentration of an analyte may require only several minutes. For comparison, the regular ELISA test for trace explosives detection may take up to six hours.

2. Aptamer incorporated PHASA biosensor outperforms immunoassays

[0089] Towards the PHASA biosensor of the present invention, aptamers are inherent to the initial design as too many disadvantages with antibodies employed in an immunoassay are foreseen:

a) Antibodies denature in organic solvents/ glycerol environments; b) Antibodies' decomposition makes limited shelf-lives in the dry or hydrated state;

c) Any antibody (or protein having tryptophan amino residues) has strong background fluorescence.

[0090] Aptamers have relatively small in size, can be stable in organic/ aqueous solvents, and achieve high ligand-specificity. Therefore, they are a more logical choice for bioassays than antibodies, including the fact that higher surface densities can be placed in a microliter plate well. [0091] Stilbene-immunoassays are not commercially available and applicable. Pursuing antibodies against different analytes is an issue, which restricts range of application and function of antibodies. The reasons for these are:

a) Antibodies against specific analytes of interest are not instantly and constantly available (e.g. monoclonal antibodies).

b) The production of antibodies requires biological systems.

c) In-vivo production of antibodies works only under physiological conditions.

[0092] Aptamers existing on the market are available for over 150 targets, thereby allowing the PHASA bioassay and biosensor device to be quickly developed towards numerous applications. Should a particular aptamer be unavailable, it can be easily designed and manufactured in 6-8 weeks for a reasonable cost, which would be unfeasible (if not impossible) for antibodies.

3. PHASA is a simple, one-step procedure

[0093] A key design feature in the PHASA platform is based on the 'no- separation-needed' concept— reagents and bound aptamers will require no discrete separation protocols or washing procedures before or after measurements. It is essentially a one-step procedure that is self-contained. The bioassay can operate not only in aqueous solutions but also in a solvent mixtures, heavily contaminated heterogeneous wastes and biological media collected in a single tube, without separate purification protocols. Eventually, the PHASA bioassay permits not only the instant detection of an analyte in a sample but also rapid determination of its concentration.

[0094] Various aspects of the various embodiments disclosed herein are combinable with the other embodiments disclosed herein. Devices, systems, structures, functionalities and methods as described herein may have other configurations, sets of components and processes etc. It should be also noted that while a device, system and method in accordance with some embodiments of the invention may be used, for example, in testing biological liquids, wastewater, solid surfaces, from hands ets, the invention is not limited in this respect. For example, some embodiments of the invention may be used in conjunction with testing sediments, air etc.

[0095] A device, system and method in accordance with some embodiments of the invention may be used, for example, in conjunction with a device which may be inserted into a human body. However, the scope of the present invention is not limited in this regard.

[0096] While certain features of the invention have been illustrated and described herein, many modifications, substitutions, changes, and equivalents will now occur to those of ordinary skill in the art. It is, therefore, to be understood that the appended claims are intended to cover all such modifications and changes as fall within the true spirit of the invention.

Related Prior Art

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Claims

CLAIMS What is claimed is:

1. A universal bioassay or biosensing device comprising:

a single aptamer or plurality of different aptamers for sensing a single analyte or plurality of different analytes, respectively; and

a switchable reporter molecule, wherein said molecule is any photochrome or fluorochrome probe capable of changing its molecular configuration upon irradiation with the UV-VIS light, and said molecule is capable of forming a stable complex with said aptamer.

2. The device according to claim 1, characterised in that said aptamer or aptamers are immobilised onto its surface.

3. The device according to claim 1, characterised in that its surface is pre-coated with metal layer or metal particles.

4. The device according to claim 3, characterised in that said metal layer or metal particles comprises:

a switchable reporter molecule, chemically bound to or immobilised onto said metal layer or metal particles, wherein said molecule is any photochrome or fluorochrome probe capable of changing its molecular configuration upon irradiation with the UV-VIS light; and

a single aptamer or plurality of different aptamers for sensing a single analyte or plurality of different analytes, respectively, wherein said aptamer or aptamers are conjugated to said switchable reporter molecule, forming a stable complex.

5. The device according to any one of claims 1-4, characterised in that said device is an optical, acoustic or electrochemical biosensor, biochip, microfluidic chip known as lab-on-chip, magnetic particles, nanostructured surfaces, for example nanoantennas, or any other bioassay or biosensing platform.

6. The device according to any one of claims 1-4, characterised in that said device is a lateral-flow strip.

7. The device according to any one of claims 1-4, characterised in that said device is a microtiter plate pre-treated with reagents for use in the commercial fluorescence microtiter plate reader.

8. The microtiter plate according to claim 5, wherein said plate is black, white or have a transparent portion.

9. The device according to any one of claims 1-4, wherein said switchable reporter molecule comprises the photochrome or fluorochrome moiety and pseudo-hapten moiety covalently bound to each other.

10. The device according to claim 9, wherein said photochrome or fluorochrome moiety is a frans-sufbene, irans-azobenzene or £ratts-l,2-bis(4-pyridyl)- ethylene derivatives.

11. The device according to any one of claims 9 and 10, wherein said pseudo- hapten moiety mimics an active part of the preselected ligand or analyte, thereby creating strong interactions with the binding site of the aptamer.

12. The device according to claim 11, wherein said preselected ligand or analyte is a small organic molecule, for example aniline and its carboxyl derivatives for serological studies, trinitrotoluene and picrates for forensic diagnostics, heroin and cocaine for drug detection and monitoring, pollutants including pesticides and toxins or any polypeptide of interests, signal toxins, for example aflatoxin, parasitic shellfish poisoning toxins (PSP), and endocrine disrupting compounds, for example Bisphenol A.

13. The device according to claim 11, wherein said preselected ligand or analyte is a protein or virus.

14. The device according to any one of claims 1-4, characterised in that said switchable reporter molecule is capable of changing its molecular configuration attaining certain isomeric form, when it is irradiated with UV- VIS light at the excitation maximum wavelength of said photochrome or fluorochrome probe.

15. The device according to claim 14, wherein said switchable reporter molecule is the fluorescent frans-stilbene derivative, which is able to undergo geometrical changes in the excited state, and consequently, adopt the non- fluorescenf cis-form.

16. The device according to claim 15, wherein said frans-stilbene derivative is:

17. The device according to claim 15, wherein said frans-stilbene derivative is:

18. The device according to claim 15, wherein said frans-stilbene derivative is:

19. The device according to any one of claims 1-4, characterised in that said device further comprises a time measuring element, which allows using the standardized calibration time for automatic performance of measurements and automatic data tracking.

20. The device according to claim 19, wherein said time measuring element may be any type of digital or analogue clocks for performing the diagnostic tests in a specific interval of time.

21. The device according to any one of claims 1-4, characterised in that said device further comprises a fluorescence or UV-VIS detector, such as photodiode, photodiode array or photomultiplier tube.

22. A universal bioassay or biosensing system comprising:

a universal biosensing device;

an external detecting instrument, which is able to detect fluorescence or spectrophotometric changes, for example fluorescence microtiter plate reader or fibre optic spectrophotometer; and

a computing platform or workstation able to store, process, display, or analyse the received data.

23. A diagnostic method based on the photochrome-aptamer switch assay, comprising the following steps:

collecting the sample from surroundings and human body, such as wastewater, body fluids, sediment, solid surfaces, skin or hands;

performing the assay using the universal bioassay or biosensing device for the predetermined period of time; and

acquiring data, such as fluorescence decay curves or intensity data, of the bioassay or biosensing device.

24. The method according to claim 23, wherein said method further comprises the step of transmitting the acquired data, analysing the data, and/ or other suitable operations.

25. A method for calibration of the bioassay or biosensing device according to any one of claims 1-4, said method is based on direct correlation of the photoisomerisation rate constant of the reporter molecule to the analyte concentration, and comprises the following steps:

steady-state fluorescence measurements of the reporter molecule in solution and in a complex with an aptamer in order to obtain two limit (end) points of the calibration curve for the complexed state and uncomplexed state (free in solution) of the reporter molecule; introducing the sample analyte of different known concentrations to achieve the competitive formation of the complex aptamer-analyte monitored by steady-state fluorescence kinetics of the reporter molecule;

calculation of the first-order trans-cis photoisomerisation rate constant for both the uncomplexed and complexed reporter molecule, and for different known concentration of the tested analyte; and

calibration of the system with the trans-cis photoisomerisation rate constant as a function of the analyte concentration at arbitrary fixed time, defined as a calibration time for field tests.

26. The method according to claim 25, wherein said method further comprises the compilation and building of a calibration database or data library for testing different analytes.

27. A method for manufacturing of the biosensing device according to claim 2, said method comprises the following steps:

surface modification of a transducer element or solid support of said biosensing device, for example quartz fibres of optical biosensors, electrodes of amperometric biosensors, metal surfaces of acoustic sensors, and polymeric surface of microliter plates or lateral flow strips, by treatment with the suitable activating, cross-linking and conjugating reagents;

coating of said transducer element or solid support of a biosensing device with the selected aptamers;

addition of the reporter molecules to said surface of the transducer element or solid support coated with the aptamer to form the complex of the reporter molecules with said aptamers; and

washing the prepared biosensing platform with buffers and organic solvents and drying in vacuum.

28. A method for manufacturing of the biosensing device according to any one of claims 3 and 4, said method comprises the following steps:

surface coating of a transducer element or solid support of said biosensing device, for example quartz fibres of optical biosensors, electrodes of amperometric biosensors, and polymeric surface of microtiter plates or lateral flow strips with metal layer or metal particles;

immobilisation of suitable activating, cross-linking or conjugating reagents onto said metal layer or metal particles;

binding or conjugating the reporter molecules to said surface of the transducer element or solid support coated with said metal layer or metal particles via said activating, cross-linking or conjugating agents;

addition of said suitable aptamer or aptamers to form the complex of the reporter molecules with them; and

29. The method according to claim 27, wherein said transducer element or solid support of the same biosensing device is coated with different aptamers for simultaneous sensing of different analytes.

30. The method according to claim 28, wherein said biosensing device comprises different aptamers for simultaneous sensing of different analytes.

31. The device according to any one of claims 1-4, characterised in that said device is fully autonomous and self-contained.

32. The device according to claim 31, characterised in that said device is a microfluidic chip or lab-on-chip, wherein all the components are substantially contained within the chip, and where the device is wireless.

33. The device according to any one of claims 1-4, characterised in that said device is a lateral flow strip, wherein the preselected aptamer is immobilised over the strip surface in a capture zone (test line), and the reporter molecule is added to form a complex with the aptamer in this zone.

34. The device according to claim 33, characterised in that said device further comprises a dedicated sensor to measure the fluorescence intensity of the test line.

35. The device according to any one of claims 1-4, characterised in that said device further comprises a microprocessor chip that calculates or provides a quantitative determination of the fluorescence intensity changes.

36. A diagnostic kit based on the photochrome-aptamer switch assay, comprising the following components:

a universal bioassay or biosensing device with an aptamer or plurality of different aptamers for sensing a single analyte or plurality of different analytes, respectively, wherein said aptamer or aptamers are immobilised onto the surface of said bioassay or biosensing device;

a switchable reporter molecule, wherein said molecule is any photochrome or fluorochrome probe capable of changing its molecular configuration upon irradiation with the UV-VIS light, and said molecule is capable of forming a stable complex with said aptamer; and a manual for operating the device.

37. A diagnostic kit based on the photochrome-aptamer switch assay, comprising the following components:

a universal bioassay or biosensing device having a surface coated with metal layer or metal particles and a switchable reporter molecule, wherein said molecule is any photochrome or fluorochrome probe capable of changing its molecular configuration upon irradiation with the UV-VIS light, characterised in that it is chemically bound to, or immobilised onto, said metal layer and said metal particles, and is capable of forming a stable complex with the corresponding aptamer;

a single aptamer or plurality of different aptamers for sensing a single analyte or plurality of different analytes, respectively, and

a manual for operating the device.