WO2013064885A1

WO2013064885A1 - Nanostructure based method for detection and/or isolation of biomolecule

Info

Publication number: WO2013064885A1
Application number: PCT/IB2012/002200
Authority: WO
Inventors: Arockiadoss THEVASAHAYAM; Harsh VARDHAN; Kiran R. AATRE; Debiprosad ROY MAHAPATRA; Chandrasekhar Bhaskaran Nair; Leela Mana OLAPPAMANA; Manoj Narayanan MULAKKAPURATH; Shilpa Sanjeeva KAMMARADI; Madhusmitaa MISHRA; Manish PRIYADARSHI; Anil Krishna
Original assignee: Bigtec Private Limited
Priority date: 2011-11-01
Filing date: 2012-11-01
Publication date: 2013-05-10

Abstract

The present invention relates to method and a composition for detecting and/or isolating a biomolecule of interest in a sample, wherein said composition comprises a plurality of nanostructures attached with a photolytic material via a linker. The method disclosed in the present invention does not cause any damaging effect on the biomolecule. A device comprising a plurality of nanostructures attached with a photolytic material via a linker as disclosed in the present invention, a support and a substrate is also provided herein.

Description

"NANOSTRUCTURE BASED METHOD FOR DETECTION AND/OR ISOLATION OF BIOMOLECULE"

FIELD OF INVENTION

The present invention relates to a method for detection and/or isolation of biomolecule using composition comprising a composite comprising nanostructures attached with a photolytic material via a linker.

BACKGROUND OF THE INVENTION

A biosensor is an analytical device that incorporates a biological recognition element in direct spatial contact with a transduction element. Molecular recognition and signal transduction are the two major challenges in biosensor design. Current biological sensing techniques commonly rely on optical detection principles. The earliest techniques involving optical detection were perceived to be inherently complex and also required multiples steps between the actual engagement of the analyte and the generation of a signal. The techniques usually also involve multiple reagents, preparative steps, signal amplification, complex data analysis and/or relatively large sample size.

Nanotechnology is emerging as a major discipline that is driving applications in a broad spectrum of fields and is making its presence felt in electronics, optical devices, industries, diagnostics, drug delivery, biosensing, imaging, as well as a variety of consumer products. The technology uses highly innovative approaches through diverse strategies and architectures for overcoming the challenges of the conventional optical, • biochemical and biophysical methods. One-dimensional nanostructured materials ( 1 D- NS), such as nanowires, nanotubes and nanorods by virtue of their small size, high sensitivity attributed to high surface-to-volume ratio, real time detection, ultra-low power demands, large surface area, near one-dimensionality of electronic transport, and potential for high-throughput and multiplexed detection are promising candidates for electronic detection of chemical and biological species. A nanostructure is an article having at least one spatial dimension of less than 1 micron. Nanostructured material also offer other significant advantages, such as new sensing mechanisms, high spatial resolution for localized detection, facile integration with standard wafer-scale semiconductor processing and label-free detection in a nondestructive manner. The technology can therefore be used for selective molecular recognition and isolation of biomolecules.

Examples of nanostructures include nanocages, nanocrystallites, nanobelts, nanoneedles, nanocomposites, nanofabrics, nanofibers, nanoflakes, nanoflowers, nanofoams, nanomeshes, nanoparticles, nanopillars, nanopin films, nanorings, nanorods, nanoshells, nanopowders, nanoclusters, nanowires, nanotubes, quantum dots, and quantum heterostructures.

There are mainly two sensing methodologies that are relevant to 1 D-NS based detection systems, namely electrical and optical. Electrical methods rely on the use of an electrical measurement as a sensor signal. A simple electrical is a resistor and a sensor that uses resistance as the sensor signal is often termed a chemiresistor. Due to the improved surface-to-volume ratio of 1 D-NS elements, this configuration is highly suited for the realization of biosensors. Other similar methods include the measurement of capacitance or impedance of the device and employing affinity based detection. Optical methodologies utilize the 1 D-NS as a marker (such as carbon nanotubes) or as a system (such as nanochannels). Typically, zero-dimensional nanostructures such as quantum dots are used as labels in bioanalytical applications.

Certain types of nanostructures that have found increasing incorporation into technologies include carbon-based nanostructures, such as carbon nanotubes and fullerenes; metal-based nanostructures, such as gold nanoparticles; and metal oxide- based nanostructures, such as Zinc oxide nanostructures and Titanium Oxide nanostructures.

One-dimensional nanostructured oxides have received ever increasing attention due to their intriguing properties and unique applications in novel nanodevices. Among them, zinc oxide (ZnO) nanostructures due to their stable and cost effective nature have found promising applications. Biocompatibility, high stability under ambient, oxygen rich physiological conditions has made the use of ZnO nanostructures increasingly popular in the fabrication of biosensors.

US201 1/0257033 describes a composition comprising a photoluminiscent nanostructure with a polymer adsorbed on the surface of the nanostructure for selective molecular recognition. The polymer may be a polysaccharide or polynucleotide.

US2004/0132070 describes a nanotube based electronic detection device for detection of protein-protein binding. The nanotube structure has a coating of electron donating polymer on the surface and has a receptor compound bound to the polymer. Niepelt et al. (Raphael Niepelt, Ulrich C Schroder, Jana Sommerfeld, Irma Slowik, Beltina Rudolph, Robert Moller, Barbara Seise, Andrea Csaki, Wolfgang Fritzsche and Carsten Ronning. Biofunctionalization of zinc oxide nanowires for DNA sensory applications, Nanoscale Research Letters, 201 1 , 6:51 1 ) describes biofunctionalization of zinc oxide (ZnO) nanowires for the attachment of DNA target molecules on the nanowire surface. With the organosilane glycidyloxypropyltrimethoxysilane acting as a bifunctional linker, amino-modified capture molecule oligonucleotides were immobilized on the nanowire surface.

However, reports suggesting the genotoxic and cytotoxic effects of ZnO nanoparticles on mammalian cells have surfaced. Sharma et al. (2009) describe the genotoxicity of ZnO. The document discloses that ZnO nanoparticles induced oxidative stress in cells which was in dicated by depletion of glutathione; catalase and superoxide dismutase. The document further discloses that ZnO nanoparticles have DNA damaging potential.

Further, when ZnO nanostructure is directly attached to DNA, the DNA molecule wraps around the nanostructure. This leads to difficulty in cleaving and isolation of the DNA molecule. Numerous nanostructure compositions are available in the art for selective molecular detection of both chemical and biological molecules incorporating either electrical or optical sensing methodology. However, there persists a need in the art for development and use of a technology that not only helps overcome the drawbacks and technical limitations of its traditional counterparts but is also easy and enables one to isolate the biomolecule per se. The methods and devices disclosed herein enable easy and less time consuming detection as. well as isolation of the biomolecule using the embodiments disclosed in the application. The nanostructures are biocompatible and exhibit a high degree of sensitivity for detection and/or isolation of a biomolecule.

SUMMARY OF THE INVNETION

An aspect of the present invention relates to a method for detecting and/or isolating a biomolecule of interest in a sample, wherein said method comprises contacting a sample comprising a biomolecule with a composite comprising a nanostructure attached with a photolytic material via a linker; detecting presence of composite-biomolecule complex formed, and isolating biomolecule from said composite-biomolecule complex. Another aspect of the present invention relates to a composition for detecting a biomolecule of interest in a sample, wherein the composition comprises a plurality of nanostructures attached with a photolytic material via a linker.

Yet another aspect of the present invention relates to a device comprising a plurality of nanostructures attached with a photolytic material via a linker, a support and a substrate. BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS

Figure 1 shows scanning electron microscopy image of ZnO nanorods grown on Zinc thin film by hydrothermal growth with a solution containing H₂0₂ of 0.6 M concentration at a hydrothermal chamber pressure of 331 .15 MPa.

Figure 2 shows schematic diagram showing the hydrothermal setup for ZnO nanorod growth.

Figure 3 shows schematic diagram showing Growth shape of hexagonal ZnO nanorods. Figure 4 shows schematic diagram showing the wurtzite structure of ZnO.

Figure 5 shows Scanning Electron Microscopic (SEM) images of the grown nanorods with different surface to volume ratio conditions.

Figure 6 shows a chemical structure of ZnO-EDC-PPP-DNA complex, in accordance with an implementation of the present subject matter, wherein A represents Zinc Oxide (ZnO), B represents l -ethyl-3-(3-dimethylaminopropyI) (EDC), C represents Protoporphyrin (PPP) and D represents DNA.

Figure 7 shows FTIR spectra of ZnO nanorods modification process: spectra marked Step (1 ) inidicates ZnO nanorod absorbance peaks. The results confirm successful functionalization of ZnO nanorods toward DNA binding. Figure 8 shows Graph representing the UV spectrophotometric analysis of different concentration of ZnO nanorods used in functionalization.

Figure 9 shows Graph representing the optimization of time for enhanced functionalization of ZnO nanorods at a concentration of 2mg/ml.

Figure 10 shows FTIR spectra of ZnO nanorods modification process: (a) step ( 1 ) (bottom, black line), functionalized ZnO nanorods (b) step (2) PPP (3C^g) bound to functionalized nanorods (red line), (c) Step (3) immobilization of ssDNA ( Ι μΜ) on the functionalized nanorod (fZnO-PPP) (blue line).

Figure 1 1 shows Graph representing the UV-Vis spectrophotometric analysis of different concentration of PPP used in binding to functionalized ZnO nanorods. The peak demarcated between dashed lines represents the one for protoporphyrin (PPP). Results confirm successful binding of PPP for increasing concentrations.

Figure 12 shows FTIR spectra of ZnO nanorods modification process: (a) step (1 ) (bottom, black line), PDHA bound to ZnO nanorods (b) step (2) PPP bound to DNA (red line), (c) Step (3) immobilization with ssDNA (blue line). Absorbance peak in step (3 ) confirm successful binding of DNA on functionalized ZnO nanorods. Figure 13 shows The UV-spectrophotometric analysis demonstrating the DNA binding to f-ZnO-PPP followed by photocleavage of ZnO-PPP-DNA complex exposed to LED (wavelength 625 run) for 2 minutes. The extent of binding was checked with 3(^g/ml of PPP and different concentration of DNA i.e. 0.2μΜ/ιη1 [Fig 13(a)], 0.5μΜ/π.1 [Fig 13(b)], Ι μΜ/ml [Fig 13(c)], 1.5μΜ/πι1 [Fig 13(d)], and 2μΜ/ιη1 [Fig 13(e)], The optimization of binding time was investigated from 1 minute to 5 minutes. Photocleavage was analyzed post 2 minutes of LED (625 run wavelength) exposure at 8001ux light intensity. The region demarcated by the dotted lines represents the peak denoting the ZnO-EDC-PPP-DNA complex and post cleavage spectroscopic properties. The peaks associated with post-cleavage marks indicate successful cleavage of DNA from the functional ized ZnO nanorods.

Figure 14 shows The Scanning Electron Micrograph depicting the ZnO nanorods anchored on the matrix of a nano composite polymer.

DETAILED DESCRIPTION OF THE INVENTION

Persons skilled in the art will be aware that the invention described herein is subject to variations and modifications other than those specifically described. It is to be understood that the invention described herein includes all such variations and modifications. The invention also includes all such steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any two or more of said steps or features.

Definitions

For convenience, before further description of the present invention, certain terms employed in the specification, examples and appended claims are collected here. These definitions should be read in light of the remainder of the disclosure and understood as by a person of skill in the art. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by a person of ordinary skill in the art. The articles "a", "an" and "the" are used to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article.

The terms "comprise" and "comprising" are used in the inclusive, open sense, meaning that additional elements may be included. It is not intended to be construed as "consists of only".

Throughout this specification, unless the context requires otherwise the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated element or step or group of element or steps but not the exclusion of any other element or step or group of element or steps.

The term "including" is used to mean "including but not limited to". "Including" and "including but not limited to" are used interchangeably.

The term "peptide" refers to two or more amino acids joined to each other by peptide bonds or modified peptide bonds. Peptides include those modified either by natural processes, such as processing and other post-translational modifications, but also chemical modification techniques. The modifications can occur anywhere in a peptide, including the peptide backbone, the amino acid side chain, and the amino or carboxyl terminal. Examples of modifications include but are not limited to amidation, acylation, acetylation, cross linking, cyclization, glycosylation, hydroxylation, phosphorylation, racemization, and covalent attachment of various moieties such as nucleotide or nucleotide derivative, lipid or lipid derivatives.

As used herein, the term "peptide" and "polypeptide" can be used interchangeably.

The term "amino acid" will refer to the basic chemical structural unit of a protein or polypeptide.

The terms "Nucleic acid," "polynucleotide", "DNA" and "nucleotide" are used interchangeably. As used herein, a "fusion protein" refers to a protein having at least two polypeptides covalently linked in which one polypeptide comes from one protein sequence or domain and the other polypeptide comes from a second protein sequence or domain.

A "gene" refers to a nucleic acid molecule whose nucleotide sequence codes for a polypeptide molecule. Genes may be uninterrupted sequences of nucleotides or they may include such intervening segments as introns, promoter regions, splicing sites and repetitive sequences. A gene can be either RNA or DNA. The R A or DNA can be single stranded or double stranded.

"Native gene" refers to a gene as found in nature with its own regulatory sequences.

"Chimeric gene" refers to any gene that is not a native gene, comprising regulatory and coding sequences that are not found together in nature. Accordingly, a chimeric gene may comprise regulatory sequences and coding sequences that are derived from different sources, or regulatory sequences and coding sequences derived from the same source, but arranged in a manner different than that found in nature.

"Synthetic genes" can be assembled from oligonucleotide building blocks that are chemically synthesized using procedures known to those skilled in the art. These building blocks are ligated and annealed to form gene segments which are then enzymatically assembled to construct the entire gene.

"Chemically synthesized", as related to a, sequence of DNA, means that the component nucleotides were assembled in-vitro. Manual chemical synthesis of DNA may be accomplished using well established procedures, or automated chemical synthesis can be performed using one of a number of commercially available machines.

The term "nanotube" refers to a hollow article having a narrow dimension (width) of about 1 to 200 nm and a long dimension (length), where the ratio of the long dimension to the narrow dimension, i.e., the aspect ratio, is at least 5. In general, the aspect ratio is between 10 and 2000. The term "nanostructure" means tubes, rods, cylinders, needles, spheres, particles, pillars, bundles, wafers, disks, sheets, plates, planes, cones, slivers, granules, ellipsoids, wedges, polymeric fibers, natural fibers, and other such objects, which have at least one spatial dimension less than about 100 nm.

The term "biomolecule" as used herein is a chemical compound that naturally occurs in living organism. Biomolecules consists primarily of carbon and hydrogen, along with nitrogen, oxygen, phosphorous and sulphur. Other elements sometimes are incorporated but are much less common.

The present invention provides a method for isolation of biomolecules using nanostructure... The nanostructures of the present invention can be carbon-based nanostructures, metal-based nanostructures or metal oxide-based nanostructures.

The examples of carbon based nanostructure include, but are not limited to carbon nanotubes and fullerenes.

The examples of metal based nanostructures include, but are not limited to Gold nanorods, Gold nanoparticles, Microgold, Gold nanowires, Gold nanospheres. Gold nanotubes.

The examples of metal oxides include, but are not limited to ZnO nanowires, ZnO nanotubes, ZnO nanopowder, ZnO nanorods, ZnO nanoneedles, ZnO nanospheres, ZnO nanoparticles, ZnO nanopillars, TiO nanowires, TiO nanotubes, TiO nanopowder, TiO nanorods, TiO nanoneedles, TiO nanospheres, TiO nanoparticles, TiO nanopillars and like.

These nanostructures can be prepared using the conventional processes known in the art for example hydrothermal method, Sol-gel method, Vapor Transport method, electrodeposition and polymer assisted growth.

There are various techniques known in the art for biomolecule separation such as chromatography, electrophoresis and centrifugation. Chromatography is the separation technique of different biomolecules based on different sizes for a mobile versus a stationary phase. The stationary phase through which mobile phase (liquid or gas) will flow, contains spherical particles packed into a column. When a mixture of sample which contain proteins are introduced into the mobile phase and allowed to pass through the column, separation takes place because the sample proteins that have a greater attraction towards the solid phase move more slowly than the sample proteins are more attracted to the mobile phase.

Several other types of techniques where the stationary phase and the substances being use for separations are possible. If their force is ionic, then separation technique is called ion exchange. Proteins of different ionic charges can be separated in this way. If substances absorb onto the stationary phase, this technique is called absorption chromatography. In gel filtration or molecular sieve chromatography, molecules are separated because of their differences in size and shape. Affinity chromatography exploits a protein's unique biochemical properties rather than the small differences

Many important biological molecules such as proteins, DNA, and RNA exist in solution as cations (+) or anions (-). Under the influence of an electric field, these molecules migrate at a rate that depends on their net charge, size and shape, the field strength, and the nature of the medium in which the molecules are moving.

Electrophoresis in biology use porous gels as the media. The sample mixture is loaded into a gel, the electric field is applied, and the molecules migrate through the gel matrix. Thus, separation is based on both the molecular sieve effect and on the electrophoretic mobility of the molecules. This method determines the size of biomolecules. It is used to separate proteins, and especially to separate DNA for identification, sequencing, or further manipulation.

Cells, debris or others intracellular materials present in solution will get separated because of centrifugal force depending on their mass, shape, or size factors. Ultra- centrifuge rotates at 600,000 is a better tool for proteins, DNA, and intracellular membranes. The present invention discloses a method of detection, isolation, purification and/or concentrating or isolating target analytes from a biological sample for example blood, sputum, urine, stool, tissue, plasma marrow etc using a combination of nanomaterials, and surfaces and photolabile compounds. The nanostructures and surfaces in fluidic channels are bound to photolabile compounds that are in turn bound to the specific target bioanalytes of interest that may be present in the biological matrix. After isolation of the target analyte a light source is used to cleave the photolabile compound thereby releasing the target analyte into a solution that can be used for further analysis.

In the present invention, ZnO nanorods were used as the matrix and were functionalized with a bifunctional carboxylic acid, 16-(2-pyridyldithiol) hexadecanoic acid (PDHA) to facilitate binding with single stranded DNA (ssDNA) which can act as probe to detect complimentary DNA from target organisms. This bifunctional acids binds to ZnO nanorods through carboxylic (COOH) functional group on one terminus and the other terminus binds to NH₂ modified ss DNA by substitution of succinimide end group (Taratula et a/.2009). This ssONA functionalized ZnO nanostructures can hybridize with complimentary DNA extracted from targeted source such as diseased organisms.

Surprisingly, it was found that nanostructures used in the composition for isolation and /or detection of a biomolecule as disclosed in the present invention does not show any gcnotoxic, cytotoxic and/or damaging side effects of the nanostructure to the biomolecule. The linker used in the method of the present invention forms a layer around the nanostructure, which does not allow the biomolecule to come in direct contact with the nanostructure. This barrier created by the presence of the liker between the nanostructure and the biomolecule enables circumventing the genotoxic, cytotoxic and damaging effects of the nanostructure to the biomolecule. The biomolecule in tum bind to the photolytic material which also makes its isolation easy and less time consuming. The biomolecules obtained are in a substantially pure form and hence can directly be used for a variety of purposes, for example in biosensors or as biological markers, without the incorporation of a further purification step. In the nanostructure based method of detection of biomolecules known in the art, the cleavage of photocleavable markers are dependent on various factors, such as photo- linker or photo-reactive moiety, wavelength of the electromagnetic excitation, and geometrical arrangement of the compositions on a binding surface. The present invention solves these problems associated with each of these aspects. In case of reported photocleavables markers, for examples, nitrobenzyle derivatives, the recovery of post-photolysis yield without side effect is degraded due to production of side products. In the present invention, no such side effect, is found as the UV-Vis spectroscopy shows only the peaks due to DNA and the elemental compositions of the composite comprising of ZnO peak, PPP peaks and no other peaks indicating that no other side products are formed. Further, IR light source is used for photo-cleaving in the present invention as opposed to UV light source used in reported photo-cleavage based bimolecular detection which may degrade protein and other biomolecules. In reported patents and literature, typical illumination times vary from 1 hour to 24 hours with yield of 1 -95%, whereas in the present invention, 100% binding and 100% photo-cleavage is confirmed while using 1 minute of illumination time, and as detected through UV-Vis spectrophotometry of pre-binding and post-cleavage test samples.

A certain distance (within about 10cm) of illumination is required according to reported literature and patents for photolytic-cleavage based biomolecular detection. In the present invention, the light exposure distance has been minimized to less than 5cm. This improvement in exposure distance is useful in compact arrangement of the detection scheme in a device. This improvement in reduced exposure distance, reduced time of illumination and low concentration of photo-lytic molecules (PPP) is also possible optimize further due to spatial arrangement of the nanostructure on supporting platform in the form of a fluidic channels in two-dimensional and three-dimensional arrays. The conventional DNA detection method based on DNA (PCR) and antibody selectivity significantly exploits the sequence specificity of the biomolecule. The sensitivity of such detection method demands purified biomolecules. For instance, the DNA extraction and purification are based on chemical, thermal and other time consuming steps followed by chromatography and other methods for purified samples. Thus, the overall detection time leads to several hours. Moreover, these methods have limited potential in field deployment, cost-effective and rapid consumption for assay.

The nanostructure based detection and/or assay system as disclosed in the present invention is capable of selective and sensitive detection of target biomolecules from a heterogenous source. For example, the cell lysate need not be processed by extensive purification steps for downstream detection. For instance, the lysate achieved from electrical lysis of cells can be incubated with the nanostructure based linker for a time period of 1 minute for DNA binding and 2 minutes for photo-cleavage based detection. A low concentration of 0.5μΜ DNA is found to be threshold for the detection. Thus, the significance of the present invention lies in fast assay technique, simple and selective process as compared to the prior art.

Traditional fluorescent based detection requires selective fluorophores which have specific excitation and emission wavelengths. The detection sensitivity of such process is limited by the time span of fluorescence emission, which is usually short in the range of 1 - 100ns. The fluorescent based detection is also affected by significant background noise caused by the non-specific fluorescence signals. Another drawback of the fluorescent based detection lies in the subsequent dye degradation during irradiation leading to weak fluorescent signal. The present invention is based on selective biomolecule binding to the nanostructure- support followed by photocleavge at the photolinker biomolecule bond. As specified in example 6, the DNA binding was achieved within 1 minute and binding stability was observed upto several minutes within 2 minutes leading to high detection sensitivity.

Several other methods of biomolecule detection are based on size based differentiation. For example, films with specific pore size were applied for separation, detection and quantification of biomolecules. However, the process has several disadvantages like leakage through pores, non uniformity in pore dimensions and dependence on biomolecule diffusion kinetics for flow through pores. The electrode based films for biomolecule affinity were limited by their strong dependence on electrode stability, conjugation chemistry and film fabrication.

The present invention overcomes the above drawbacks by simplified and stable fabrication process of nanostructures with linkers. The nanostructure-linker complex as disclosed in the present invention acts as a bed downstream to cell lysis process where a heterogeneous system of biomolecules are formed in which the target biomolecules (e.g., DNA or protein) are mixed. The DNA binding and cleavage would be detected by the photolinker-biomolecule conjugation and photolytic cleavage of the bond. Post cleavage the qualitative and quantitative assay can be followed by simplified process of UV spectrophotometry, fluorimetry, gel analysis, microscopy and others.

In an aspect of the present invention, crystalline ZnO nanorods are prepared by hydrothermal method with controlled condition of pressure, temperature and catalysts. The size, length and purity level along with morphology of the ZnO nanorod growth are optimized. The nanorods are grown on a seed layer or a substrate. The nanorods are then removed from the substrate or the seed layer for their functional ization.

For functionalization, the nanostructures are taken in powder form. 2mM 16-(2-pyridyl dithiol) hexadecanoic acid (PDHA) taken in a mixture of ethanol and butanol in the ratio of 2: 1 along with N-(15-carboxy pentadecanoyloxy) succinimide (NHS). The solution is mixed with the powdered ZnO nanostructure in Phosphate Buffer for upto 2 hours. Subsequently the mixture was washed with deionized water and dried. Finally, functionalized nanostructures designated as "f- nanostructures" were are collected in powder form. The f- nanostructures were then attached to a linker.

An example of linker includes, but are not limited to l -ethyl-3-(3- dimethylaminopropyl) (EDC), thio self assembly mono layer ( 1 ,8-octanedithiol); ( 1 ,4- bis(mercaptomethyl) benzene; l ,4-bis(mercaptomethyl) cyclohexane; 1 ,4- bis(mercaptoacetamido) benzene; l ,4-bis(mercaptoacetamido) cyclohexane and sulfhydryl. For attachment of linker, the functionalized nanostructures in powder form were mixed with a solution of N-(15-carboxy pentadecanoyloxy) succinimide (NHS) and 2-(N- morpholine)-ethane sulfonic acid (MES) in deionized water. After 5 minutes, the linker, l -ethyl-3-(3-dimethylaminopropyl) (EDC) was added and the final mixture was mixed for about 6 hours. The mixture was washed with deionized water and dried to collect nanostructure-linker in white powder form. The nanostructure-linker was attached to a photolytic material.

The examples of photolytic material include, but are not limited to Protoporphyrin (PPP), Porphyrin meso-tetrakis-[4-(carboxy methyleneoxy) phenyl] porphyrin (H2T4CPP), 6-hydroxy - l -methylene-3-oxo-3Hbenzopyrans, 6-methoxy - 1 -methylene- 3-oxo-3Hbenzopyrans, Aspartyl β-nitrobenzyl ester, chlorine peroxide molecule, 2- nitrobenzyl group, tetracationic monointercalant, cyclo-bisintercalant, 4,4-bipyridinium, oligonucleotides, oxazole yellow (YO), dimer of oxazole yellow (YOYO), dimer of thiazole orange (TOTO), cis- and trans- components.

In an implementation of the present subject matter, for attachment of photolytic material, nanostructure-linker was mixed with a solution containing N-(15-carboxy pentadecanoyloxy) succinimide (NHS), (2-(N-morpholine)-ethane sulfonic acid (MES) in deionized water. To this mixture, the photolytic material, PPP was added and the entire mixture was dissolved in Methanol. The solution mixture was stirred for about 6 hours in absence of light. Subsequently, the solution mixture was washed with deionized water and dried to collect nanostructure-linker-photolytic material complex (f- nanostructure-EDC-PPP) in the form of brown powder.

After obtaining the nanostructure-linker-photolytic material complex, biomolecules are bound to the complex by contacting the complex with a sample comprising the biomolecule.

The examples of sample include, but are not limited to a crude extract, blood, semen, saliva, urine, mucus, stool, sputum, protein mixture and any other biological sample. The examples of biomolecules include, but are not limited to amino acids, peptides and nucleic acid. The peptides include polypeptides, proteins, fusion proteins and like. The nucleic acid includes DNA or RNA. The DNA and RNA may be single stranded or double stranded. The DNA includes native genes, chimeric genes, foreign genes, synthetic genes and chemically synthesized gene. The nanostructure-linker-photolytic material complex was mi xed with NHS, (2 -(N- morpholine)-ethane sulfonic acid (MES) in deionized water to obtain mixture. The biomolecule sample was added to the mixture to obtain the nanostructure-linker- photolytic material-biomolecule complex. Binding reaction time was optimized in the range of 30 seconds to 2 minutes depending on various parameters such as the size of the biomolecule and chemistry of end terminals.

The binding of biomolecules to the complex was confirmed by the conventional methods such as microscopy, Polymerase Chain Reaction (PCR), absorbance and scattering method. The bound biomolecules were cleaved by exciting the solution containing the nanostructure-linker-photolytic material-biomolecule complex with light of suitable wavelength. The wavelength is pre-selected depending on the absorption characteristics of the photolytic material used. The light used can be a visible light or ultraviolet radiation or infrared radiation.

Certain existing reports put forth the bactericidal effect of ZnO and its inhibitorial effect on DNA amplification (Xie et al , 201 1 ). The present invention provides a method of detection of biomolecule that involves steps to overcome the toxicity effect of nanorods in the downstream screening process of biomolecules. The ZnO nanorods of the present invention were synthesized by hydrothermal effect as described in Example 1 and thereafter embedded in a polymer of nanocomposite so as to firmly anchor the nanorods and prevent leaching into the medium. This embodiment within the polymer yields a typical floral type of geometrical arrangement (Fig.14.) which exposes the nanorod tips for the step wise functionalization and binding of biomolecule. This process excludes the possibility of nanorod release into the test sample and its interfering effects in the diagnostic process.

The method of the present invention can further be used for extracting biomolecules, protein sizing and quantification, protein biomarker identification, drug discovery and disease screening. Porphyrin-peptide conjugates are reported to be used in peptide as well as porphyrin delivery into cell (Mezo et al , 201 1 ). Based on the same principle as for ZnO-DNA binding, synthetic oligopeptides can be conjugated to porphyrin molecules based on their metal cofactors and end terminal chemical bond. The ZnO backbone as disclosed in the present invention can be linked to metalloporphyrins which can selectively bind to cationic aminoacids on a peptide chain. The present invention in particular provides a method for isolation of biomolecules from a sample, the method comprises functionalizing a nanostructure to obtain a functionalized nanostructure; attaching a linker to the functionalized nanostructure; linking a photolytic material to the linker; contacting a sample having biomolecules to obtain a nanostructure-linker-photolytic material-biomolecule complex; and exposing the nanostructure-linker-photolytic material-biomolecule complex to light to isolate the biomolecule.

In an embodiment of the present invention, there is provided a method for isolation of biomolecules from a sample, the method comprises functionalizing a nanostructure to obtain a functionalized nanostructure; attaching a linker to the functionalized nanostructure; linking a photolytic material to the linker; contacting a sample having biomolecules to obtain a nanostructure-linker-photolytic material-biomolecule complex; and exposing the nanostructure-linker-photolytic material-biomolecule complex to light to isolate the biomolecule, wherein the nanostructure is selected from the group consisting of carbon nanotubes, fullerenes, Gold nanorods, Gold nanoparticles, Microgold, Gold nanovvires, Gold nanospheres, Gold nanotubes, Zinc oxide nanowires, Zinc oxide nanotubes, Zinc oxide nanopowder. Zinc oxide nanorods, Zinc oxide nanoneedles, Zinc oxide nanospheres, Zinc oxide nanoparticles , Zinc oxide nanopillars. Titanium oxide nanowires, Titanium oxide nanotubes, Titanium oxide nanopowder, Titanium oxide nanorods, Titanium oxide nanoneedles, Titanium oxide nanospheres, Titanium oxice nanoparticles, Titanium oxide nanopillars.

In yet another embodiment of the present invention, there is provided a method for isolation of biomolecules from a sample, the method comprises functionalizing a nanostructure to obtain a functionalized nanostructure; attaching a linker to the functionalized nanostructure; linking a photolytic material to the linker; contacting a sample having biomolecules to obtain a nanostructure-linker-photolytic material- biomolecule complex; and exposing the nanostructure-linker-photolytic material- biomolecule complex to light to isolate the biomolecule, wherein the nanostructure is Zinc oxide nanorods.

In another embodiment of the present invention, there is provided a method for isolation of biomolecules from a sample, the method comprises functionalizing a nanostructure to obtain a functionalized nanostructure; attaching a linker to the functionalized nanostructure; linking a photolytic material to the linker; contacting a sample having biomolecules to obtain a nanostructure-Iinker-photolytic material-biomolecule complex; and exposing the nanostructure-linker-photolytic material-biomolecule complex to light to isolate the biomolecule, wherein the linker is selected from the group consisting of 1 - ethyl-3-(3-dimethylaminopropyl) (EDC), thio self assembly mono layer ( 1 ,8- octanedithiol); ( l ,4-bis(mercaptomethyl)benzene; l ,4-bis(mercaptomethyl)cyclohexane; l ,4-bis(mercaptoacetamido)benzene; 1 ,4-bis(mercaptoacetamido) cyclohexane) and sulfhydryl or combination thereof.

In still another embodiment of the present invention, there is provided a method for isolation of biomolecules from a sample, the method comprises functionalizing a nanostructure to obtain a functionalized nanostructure; attaching a linker to the functionalized nanostructure; linking a photolytic material to the linker; contacting a sample having biomolecules to obtain a nanostructure-linker-photolytic material- biomolecule complex; and exposing the nanostructure-linker-photolytic material- biomolecule complex to light to isolate the biomolecule, wherein the linker is l -ethyl-3- (3-dimethylaminopropyl) (EDC).

In yet another embodiment of the present invention, there is provided a method for isolation of biomolecules from a sample, the method comprises functionalizing a nanostructure to obtain a functionalized nanostructure; attaching a linker to the functionalized nanostructure; linking a photolytic material to the linker; contacting a sample having biomolecules to obtain a nanostructure-linker-photolytic material- biomolecule complex; and exposing the nanostructure-linker-photolytic material- biomolecule complex to light to isolate the biomolecule, wherein the photolytic material is selected from the group consisting of Protoporphyrin (PPP), Porphyrin meso-tetrakis- [4-(carboxy methyleneoxy) phenyl] porphyrin (H2T4CPP), 6-hydroxy - l -methylene-3- oxo-3Hbenzopyrans, 6-methoxy - l -methylene-3-oxo-3Hbenzopyrans, Aspartyl β- nitrobenzyl ester, chlorine peroxide molecule, 2-nitrobenzyl group, tetracationic monointercalant, cyclo-bisintercalant, 4,4-bipyridinium, oligonucleotides, oxazole yellow (YO), dimer of oxazole yellow (YOYO), dimer of thiazole orange (TOTO), cis- and trans- components.

In another embodiment of the present invention, there is provided a method for isolation of biomolecules from a sample, the method comprises functionalizing a nanostructure to obtain a functionalized nanostructure; attaching a linker to the functionalized nanostructure; linking a photolytic material to the linker; contacting a sample having biomolecules to obtain a nanostructure-linker-photolytic material-biomolecule complex; and exposing the nanostructure-linker-photolytic material-biomolecule complex to light to isolate the biomolecule, wherein the photolytic material is Protoporphyrin (PPP).

In still another embodiment of the present invention, there is provided a method for isolation of biomolecules from a sample, the method comprises functionalizing a nanostructure to obtain a functionalized nanostructure; attaching a linker to the functionalized nanostructure; linking a photolytic material to the linker; contacting a sample having biomolecules to obtain a nanostructure-linker-photolytic material- biomolecule complex; and exposing the nanostructure-linker-photolytic material- biomolecule complex to light to isolate the biomolecule, wherein the sample is selected from the group consisting of a crude extract, blood, semen, saliva, urine, mucus, stool, sputum, protein mixture and any other biological sample.

In an embodiment of the present invention, there is provided a method for isolation of biomolecules from a sample, the method comprises functionalizing a nanostructure to obtain a functionalized nanostructure; attaching a linker to the functionalized nanostructure; linking a photolytic material to the linker; contacting a sample having biomolecules to obtain a nanostructure-linker-photolytic material-biomolecule complex; and exposing the nanostructure-linker-photolytic material-biomolecule complex to light to isolate the biomolecule, wherein the biomolecule is selected from the _/group consisting of amino acids, peptides and nucleic acid.

In yet another embodiment of the present invention, there is provided a method for isolation of biomolecules from a sample, the method comprises functionalizing a nanostructure to obtain a functionalized nanostructure; attaching a linker to the functionalized nanostructure; linking a photolytic material to the linker; contacting a sample having biomolecules to obtain a nanostructure-linker-photolytic material- biomolecule complex; and exposing the nanostructure-linker-photolytic material- biomolecule complex to light to isolate the biomolecule, wherein the biomolecule is selected from the group consisting of peptides, polypeptides, proteins, fusion proteins, single stranded RNA, double stranded RNA, single stranded DNA and double stranded DNA.

In still another embodiment of the present invention, there is provided a method for isolation of biomolecules from a sample, the method comprises functionalizing a nanostructure to obtain a functionalized nanostructure; attaching a linker to the functionalized nanostructure; linking a photolytic material to the linker; contacting a sample having biomolecules to obtain a nanostructure-linker-photolytic material- biomolecule complex; and exposing the nanostructure-linker-photolytic material- biomolecule complex to light to isolate the biomolecule, wherein the biomolecule is a double stranded D A.

In another embodiment of the present invention, there is provided a method for isolation of biomolecules from a sample, the method comprises functionalizing a nanostaicture to obtain a functionalized nanostructure; attaching a linker to the functionalized nanostructure; linking a photolytic material to the linker; contacting a sample having biomolecules to obtain a nanostructure-linker-photolytic material-biomolecule complex; and exposing the nanostructure-linker-photolytic material-biomolecule complex to light to isolate the biomolecule, wherein the light is a visible light or ultraviolet radiation or infrared radiation. In an embodiment of the present invention, there is provided a method for isolation of biomolecules from a sample, the method comprises functionalizing a nanostructure to obtain a functionalized nanostructure; attaching a linker to the functionalized nanostructure; linking a photolytic material to the linker; contacting a sample having biomolecules to obtain a nanostructure-linker-photolytic material-biomolecule complex; and exposing the nanostructure-linker-photolytic material-biomolecule complex to light to isolate the biomolecule, wherein the light is a visible light.

In yet another embodiment of the present invention, there is provided a method for isolation of biomolecules from a sample, the method comprises functionalizing a nanostructure to obtain a functionalized nanostructure; attaching a linker to the functionalized nanostructure; linking a photolytic material to the linker; contacting a sample having biomolecules to obtain a nanostructure-linker-photolytic material- biomolecule complex; and exposing the nanostructure-linker-photolytic material- biomolecule complex to light to isolate the biomolecule, wherein the light is an ultraviolet radiation. In still another embodiment of the present invention, there is provided a method for isolation of biomolecules from a sample, the method comprises functionalizing a nanostructure to obtain a functionalized nanostructure; attaching a linker to the functionalized nanostructure; linking a photolytic material to the linker; contacting a sample having biomolecules to obtain a nanostructure-linker-photolytic material- biomolecule complex; and exposing the nanostructure-Iinker-photolytic material- biomolecule complex to light to isolate the biomolecule, wherein the light is an infrared radiation. In accordance with the present invention, there is provided a method for detecting and/or isolating a biomolecule of interest in a sample, wherein the method comprises contacting a sample comprising a biomolecule with a composite comprising a nanostructure attached with a photolytic material via a linker; detecting presence of composite-biomolecule complex formed, and isolating - biomolecule from said composite-biomolecule complex.

In some embodiment, the nanostructure as disclosed in the present invention is selected from a group consisting of nanocages, nanocrystallites, nanobelts, nanoneedles, nanocomposites, nanofabrics, nanofibers, nanoflakes, nanoflowers, nanofoams, nanomeshes, nanoparticles, nanopillars, nanopin films, nanorings, nanorods, nanoshells, nanopowders, nanoclusters, nanowires, nanotubes, quantum dots, quantum heterostructures.

In some embodiments, the nanostructure as disclosed in the present invention is selected from a group consisting of carbon-based nanostructure, metal-based nanostructure, metal oxide-based nanostructure and silica based nanostructure. In some embodiments, the carbon-based nanostructure as disclosed in the present invention is selected from a group consisting of carbon nanotubes, a single-walled carbon nanotubes, multi-walled carbon nanotubes and fullerenes.

In other embodiments, the metal-based nanostructure as disclosed in the present invention is selected from a group consisting of gold, silver, nickel, platinum nanoparticles. In some embodiments, the metal oxide-based nanostructure as disclosed in the present invention is Zinc oxide nanostructure or Titanium dioxide or Iron oxide nanostructure.

In some embodiments, the biomolecule is selected from a group consisting of nucleic acid, DNA, RNA, SNP, cell marker proteins, cellular toxins, polypeptide, peptide, amino acid, protein, fusion protein, antibody, antibody fragment, small molecules, lipids and RNA or DNA virus.

In some embodiments, the sample is selected from a group consisting of crude extract, blood, semen, tears, saliva, urine, mucus, stool, sputum, phlegm, and protein mixture, sweat, urine, plasma, lymph, spinal fluid, cells, microorganisms, a combination thereof, and aqueous dilutions thereof. In another embodiment of the present invention there is provided a composition for detecting and/or isolating a biomolecule of interest in a sample, wherein the composition comprises a plurality of nanostructures attached with a photolytic material via a linker.

In further embodiment of the present invention, there is provided a device for detecting and/or isolating a biomolecule of interest in a sample comprising a plurality of nanostructures attached with a photolytic material via a linker, a support and a substrate.

Advantages of the Invention

The nanomaterial based composition as disclosed in the present invention can be used for biodiagnostic screening of proteins and nucleic acids and other relevant biomolecules. Further, nanostructured materials as disclosed in the present invention exhibits a unique electronic and photonic properties with biomaterials. The nanorods of the present invention are used for controlling electrical and catalyst effect of different biomolecule samples and isolation of intracellular membranes from a given sample. Furthermore, ^'.he nanomaterial based composition as disclosed in the present invention shows synergetic properties for binding antigen, antibody or DNA and exhibits increased binding strength and immobilizes the targeted DNA, protein or small biomolecules.

The principles and practice of the invention may contribute, in due course, to the development of sensors and to using nanoscale devices for in-vivo applications directed toward cellular physiology, medical screening, and diagnosis. Sensor devices based on the nanostructure composition as disclosed in the present invention may be constructed, according to the principles of the invention.

Having thus described embodiments of the present invention it should be apparent to those skilled in the art that certain advantages of the within system have been achieved. It should also be appreciated that various modifications, adaptations, and alternative embodiments thereof may be made within the scope and spirit of the present invention.

EXAMPLES

It should be understood that the following examples described herein are for illustrative purposes only and that various modifications or changes in light of the specification will be suggestive to person skilled in the art and are to be included within the spirit and purview of this application and the scope of the appended claims.

Example 1

Preparation of ZnO based Nanostructures

A novel method was developed based upon pressure assisted hydrothermal grovvlh of tunable ZnO single crystal nanorods structures. The pressure on nucleation site was defined by the height of the water column. Dimensions of the growing features such as length, diameter, and cluster size were optimized by a fine tuning of pressure over the nucleation site. The morphology and dimensions of the ZnO nanorods were measured and analysed. Hydrothermal method is a technique to grow single crystals in high temperature and high vapor pressure solution. Hydrothermal reaction takes place during the heating and cooling span and during this duration particle nucleates and grows. This method facilitates the dissolution of almost all inorganic substances in water at high temperatures and pressures. The dissolved material subsequently crystallizes from the fluid. The reaction was carried out in a Teflon bottle by external heating in a furnace, the bottle worked as a hydrothermal chamber.

Step 1 : Zinc substrates of dimensions 10 mm x 10 mm x 0.25mm were taken.

Step 2: Zinc foil was first cleaned by ultrasonication in acetone for 5 minutes followed by wash with distilled water to make the surface free from dust and oxide particles and any other surface adsorbents before loading for the growth process.

Step 4: Each foil was then placed at the centre of the base inside the hydrothermal chambers.

Step 5: Volume was filled up to a level in the chamber thereby producing a liquid column which exerts pressure at the base. The pressure on the base was correlated to liquid volume and the volume of the air column.

Step 6: The hydrothermal chamber was sealed with airtight screw cap and the setup was heated at 100°C for 14 hrs.

Size and Shape of Nanostructures

Nanorods of length scale 200 to 300 nm and diameter up to 90 nm was grown by the above mentioned process. In its stable wurtzite crystal form, zinc oxide crystallizes in hexagonal form which corresponds to space group C6mc. In such configuration, it has lattice parameters a = 0.325nm and c = 0.521 nm. This structure consists of planes that arc composed of tetrahedrally coordinated 0^2~ and Zn²⁺ ions. The basal plane is the most common polar plane. Fig.3 represents the typical growth shape of ID hexagonal shaped ZnO nanorod. Planar defects and twins have been found to occur parallel to the (0001 ) plane, however, dislocations are rarely observed in this crystal.

Structural Details

Zinc oxide exhibits piezoelectric properties due its unique structure. The origin of piezoelectric characteristic is because of crystalline structure in which the oxygen atoms and zinc atoms are tetrahedrally bonded. The hydrothennal technique was followed to synthesize nanorods of ZnO.

Example 2

Characterization of ZnO nanorods

The preliminary characterization of ZnO nanorods were based upon the Scanning Electron Microscopy (SEM) (Zeiss Ultra55) images. The presence of crystalline phases was determined from XRD pattern for nanorods array film prepared with distilled H₂0 and 0.6M H₂0₂ in 30 ml of the solution. Table 1 summarizes the SEM properties of the nanorods grown at various pressures in different solutions. Nanorods of length scale 200 to 300 nm and diameter up to 90 nm have been grown by the above mentioned hydrothermal scheme. Reaction enthalpy at the Zn substrate surface is expected to increase in (South Pole) direction along with an increment in hydrostatic pressure.

It was observed that at lower pressures the nanorods form needle type tips at the end point, whereas at higher pressures the rod terminals are flat in shape. The flat tips emphasize an incomplete growth of the nanostructures during the applied heating and cooling ramping time. This can be attributed to more number of OH^' ions available from reactions through deep diffusion. These hydroxyl ions require more number of zinc ions which can be provided throughout process duration higher than 14 hours, to complete the reaction and finish the nanorod formation to a pointed tip. Surprisingly no nanorod formation was observed for the sample prepared with 0.4 M H₂0₂ solution at 360 MPa. We have located few regions on the grown nanorods array film with distinct features different from rest of the nanorods distribution. The corresponding images are represented in Fig.5. These structures claim their importance in electronic and sensing applications.

Table 1 : The dimensional features of nanorods grown at various concentrations

Maximum Minimum Minimum Maximum

Solution used

Length (μηι) Length (μιη) Diameter (nm) Diameter (nm) Dist. H₂0 0.2698 0.20338 69.61 89.59

0.2 M H₂O2 0.9609 5.702 163.5 1595.0

0.4 M H₂0₂ 1.331 5.5888 362.2 1045

0.6 M H₂0₂ 0.8074 0.2359 462.5 1063.0 Example 3

Surface Functionalization of ZnO nanorods

2 mg of ZnO nanorods as described in Example 1 was taken in powder form and 2 mM of 16-(2-pyridyl dithiol) hexadecanoic acid (PDHA), 4 mM of N-(15-carboxy pentadecanoyloxy) succinimide (NHS) was added to it. 10 mM phosphate buffer was added to above mixture to make up the volume to 1 ml. Above mixture was stirred at 300 rpm for 2 hours at room temperature 2 hours. The mixture was then washed with deionized water thoroughly by centrifugation at 5000rpm for 5 minutes and dried.

Step 1 : 2 mg of ZnO nanorods in powder form of Example 1

Step 2: 2mM of bifunctional carboxylic acid 16-(2-pyridyldithiol) hexadecanoic acid (PDHA) dissolved in 2: 1 butanol: ethanol.

Step 3: 4mM NHS buffer

Step 4: l OmM phosphate buffer was added to above mixture to make up the volume to 1 ml.

Step 5: Above mixture was stirred at 300 rpm for 2 hours at room temperature.

Step 6: The above mix was washed thrice with deionized water by centrifugation at 5000 rpm for 5 minutes.

Step 7: The washed sample was dried at room temperature and functional ized Zn (f- ZnO) nanorod was collected.

Step 8: Characterization of f-ZnO was done UV-Vis and FT-IR spectroscopy Charcterization ZnO functionalization was done by UV spectroscopy. As represented in Fig.8 increasing concentration of ZnO gives a linear yield in functionalized ZnO nanorods by the above optimized process. With a saturated concentration of 2mg/ml ZnO nanorods, functionalization time was optimized and Fig.9 clearly presents that incubation time span of 2 hours results in high yield of functionalized ZnO nanorods.

Example 4

Linker attachment on the f-ZnO nanorods 2 mg of f-ZnO nanorods prepared by the process as described above were added to 50 mM of N-( l 5-carboxy pentadecanoyloxy) succinimide (NHS) and 50 mM of 2-(N- morpholine)-ethane sulfonic acid (MES) in 10 ml deionized water. 50 mM of l -ethyl-3- (3-dimethylaminopropyl) (EDC) was added to above suspension and the mixture was stirred for up to 6 hours at room temperature. The mixture was thoroughly washed with deionized wa^*er by centrifugation at 5000 for 5minutes and thereafter dried to collect ZnO-EDC.

Step 1 : 2mg of f-ZnO nanorods of Example 3

Step 2: 50mM of N-hydroxysuccinimide (NHS) buffer and 50mM of 2-(N-morpholino) ethanesulfonic acid (MES) buffer in 10ml distill water.

Step 3: 50mM of l -ethyl-3- 3-dimethylaminopropyl carbodiimide (EDC) was added to the above mix followed by stirring at 300 rpm for 6 hours at room temperature.

Step 4: The above mix was washed thrice with deionized water by centrifugation at 5000 rpm for 5 minutes.

Step 5: The washed sample was dried at room temperature and f-ZnO-EDC was collected.

Example 5

Attachment of Photolytic Material on the Linker

ZnO-EDC collected from above step was added to a solution containing 50 mM of N- (15-carboxy pentadecanoyloxy) succinimide (NHS) and 50 mM of (2-(N-morpholine)- ethane sulfonic acid (MES) in 10ml deionized water. 2 mg of photolytic material Protoporphyrin (PPP) was added to the above solution and the entire mixture was dissolved in 3 ml of Methanol. The mixture was stirred for up to 6 hours in absence of light at room temperature and thereafter the mixture was washed with deionized water to collect ZnO-EDC-PPP.

Step 1: f-ZnO-EDC collected from Example 4

Step 2: 2 mg of a photolytic material Protoporphyrin IX (PPP) in 3 ml of methanol. Step 3: 50mM of NHS buffer and 50mM of MES buffer in 10ml distill water.

Step 4: Above suspension was stirred for up to 6 hours in absence of light at 300 rpm under room temperature.

Step 5: The above mix was washed with deionized water by centrifugation at 5000 rpm for 5 minutes.

Step 6: ZnO-EDC-PPP was collected and the complex formation was confirmed by UV Vis as well as FT-IR spectroscopy

The binding of photolytic material i.e. PPP to the f-ZnO-EDC complex was analyzed by UV spectroscopy method. Different concentration of PPP was added to the ZnO-EDC complex to optimize the efficient binding. Based on UV spectroscopy results (Fig.1 1.), 30 (.ig of PPP concentration was used for the DNA binding analysis. Example 6

Isolation of DNA molecules using the ZnO nanorods by Photo-cleavage method

ZnO-EDC-PPP as prepared in Example 5 was added in 50 mM of NHS and 50 mM of (2-(N-morpholine)-ethane sulfonic acid (MES) in 10ml of deionized water. DNA binding to above complex was checked with different concentration of DNA ranging from 0.2-2μΜ. The extent of binding was checked over a time span of 1 to 5 minute, and the binding was confirmed by UV spectroscopy. Fig.6. represents the schematic diagram of ZnO-EDC-PPP-DNA complex, where in A represents Zinc Oxide (ZnO), B represents l-ethyl-3-(3-dimethylaminopropyl) (EDC), C represents Protoporphyrin (PPP) and D represents DNA. For photoclea /age, the solution containing ZnO-EDC-PPP-DNA was excited with LED of wavelength ~650nm for up to 2 minutes. Step 1: ZnO-EDC-PPP of Example 5 was added in 50 mM of NHS and 50 mM of MES buffer in 10ml of DI water.

Step 2: 0.2-2μΜ DNA was added to above mixture (Step 20) and incubated for upto 5 minutes.

Step 3: At regular time intervals, DNA binding was analyzed by UV spectrophotometry and FTIR spectroscopy

Step 4: The suspension having DNA bound to PPP was exposed to LED (goo650nm wavelength) for up to 2 min.

Step 5: The cleavage was checked under different intensities and for different time of exposure.

Step 6: DNA cleavage was analyzed by UV spectrophotometry

DNA binding and photocleavage was analyzed by UV spectrophotometry. DNA binding to ZnO-EDC-PPP leads to a complex structure formation which results an increase in UV absorbance at 260nm as compared to the same concentration of free DNA added to buffer. For the lowest concentration of DNA, 0.2μΜ, no binding was observed inspite of incubation till 4 minutes [Fig 13(a)j.As the DNA concentration was increased the time required for complex formation increases as observed in case of 1 2μΜ DNA [Fig 13(d) and Fig 13(e)]. The photocleavgae was indicated by the drastic drop in UV absorbance which was similar to the absorbance yielded by free DNA in buffer. Post cleavage, UV absorbance was measured post incubation of samples for 5 minutes at room temperature so as to prevent chances of reannealing and confirm complete cleavage.

The UV spectrophotometric and FT-IR analyses also indicate a selective binding of the biomolecule, i.e. binding of DNA to the functionalized nanorod with photo-linker. The attachment of the DNA probe to the PPP leads to an increase in the UV absorbance as compared to either the unbound PPP or the free DNA in the buffer (Fig.13). The selective binding of the nanorod-biomolecule is also reflected by the concentration dependent factor of biomolecule for optimal binding. As depicted in Fig 13(a) to 13(e), an optimal concentration of biomolecule, for example, at concentration above 1 μΜ, was required for the complex formation. The step-wise functionalization followed by subsequent binding of the photolinker and specific biomolecule was also confirmed by FT-IR analysis (step 1 -3 of Fig.12.). The present technique of nanorod based screening of biomolecules holds the advantage of being a simple and efficient and fast process of functionalization and biomolecule binding.

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Claims

IAVe Claim:

1. A method for detecting and/or isolating a biomolecule of interest in a sample, wherein said method comprises contacting a sample comprising a biomolecule with a composite comprising a nanostructure attached with a photolytic material via a linker; detecting presence of composite-biomolecule complex formed, and isolating biomolecule from said composite-biomolecule complex.

2. The method as claimed in claim 1 , wherein the nanostructure is selected from a group consisting of nanocages, nanocrystallites, nanobelts, nanoneedles, nanocomposites, nanofabrics, nanofibers, nanoflakes, nanoflowers, nanofoams, nanomeshes, nanoparticles, nanopillars, nanopin films, nanorings, nanorods, nanoshells, nanopowders, nanoclusters, nanowires, nanotubes, quantum dots, quantum heterostructures.

3. The method as claimed in claim 1 , wherein the nanostructure is selected from a group consisting of carbon-based nanostructure, metal-based nanostructure, metal oxide-based nanostructure and silica based nanostructure.

4. The method as claimed in claim 3, wherein the carbon-based nanostructure is selected from a group consisting of carbon nanotubes, a single-walled carbon nanotubes, multi-walled carbon nanotubes and fullerenes.

5. The method as claimed in claim 3, wherein the metal-based nanostructure is selected from a group consisting of gold, silver, platinum, nickel nanoparticles.

6. The method as claimed in claim 3, wherein the metal oxide-based nanostructure is Zinc oxide nanostructure or Titanium dioxide nanostructure or Iron oxide.

7. The method as claimed in claim 1 , wherein the photolytic material is selected from a group consisting of Protoporphyrin (PPP), Porphyrin meso-tetrakis-[4-(carboxy methyleneoxy) phenyl] porphyrin (H2T4CPP), 6-hydroxy -l -methylene-3-oxo- 3Hbenzopyrans, 6-methoxy - l-methylene-3-oxo-3Hbenzopyrans, Nitrobenzyl,

Aspartyl β-nitrobenzyl ester, chlorine peroxide molecule, 2-nitrobenzyl group, tetracationic monointercalant, cyclo-bisintercalant, 4,4-bipyridinium, oligonucleotides, oxazole yellow (YO), dimer of oxazole yellow (YOYO), dimer of thiazole orange (TOTO), cis- and trans- components.

8. The method as claimed in claim 1 , wherein the linker is selected from a group consisting of l -ethyl-3-(3-dimethylaminopropyl) (EDC), thio self assembly mono layer (1 ,8-octanedithiol); l ,4-bis(mercaptomethyl) benzene; 1 ,4- bis(mercaptomethyl) cyclohexane; 1 ,4-bis (mercaptoacetamido) benzene; 1 ,4- bis(mercaptoacetamido) cyclohexane and sulfhydryl.

9. The method as claimed in claim 1 , wherein the biomolecule is selected from a group consisting of nucleic acid, DNA, RNA, SNP, cell marker proteins, cellular toxins, polypeptide, peptide, amino acid, protein, fusion protein, antibody, antibody fragment, and RNA or DNA virus.

10. The method as claimed in claim 1 , wherein the sample is selected from a group consisting of crude extract, blood, semen, tears, saliva, urine, mucus, stool, sputum, phlegm, and protein mixture, sweat, urine, plasma, lymph, spinal fluid, cells, microorganisms, a combination thereof, and aqueous dilutions thereof.

1 1. A composition for detecting and/or isolating a biomolecule of interest in a sample, wherein said composition comprises a plurality of nanostructures attached with a photolytic material via a linker.

12. A device for detecting and/or isolating a biomolecule of interest in a sample comprising a plurality of nanostructures attached with a photolytic material via a linker, a support and a substrate.

13. The composition as claimed in claim I I or the device as claimed in claim 12, wherein the nanostructure is selected from a group consisting of nanocages, nanocrystallites, nanobelts, nanoneedles, nanocomposites, nanofabrics, nanofibers, nanoflakes, nanoflowers, nanofoams, nanomeshes, nanoparticles, nanopillars, nanopin films, nanorings, nanorods, nanoshells, nanopowders, nanoclusters, nanowires, nanotubes, quantum dots, quantum heterostructures.

14. The composition as claimed in claim 1 1 or the device as claimed in claim 12, wherein the nanostructure is selected from a group consisting of carbon-based nanostructure, metal-based nanostructure, metal oxide-based nanostructure and silica based nanostructure.

1 5. The composition or the device as claimed in claim 13, wherein the carbon-based nanostructure is selected from a group consisting of carbon nanotubes, a single- walled carbon nanotube, multi-walled carbon nanotube and fullerenes.

16. The composition or the device as claimed in claim 13, wherein the metal-based nanostructure is selected from a group consisting of gold, silver, nickel, platinum nanoparticles.

17. The composition or the device as claimed in claim 13, wherein the metal oxide- based nanostructure is Zinc oxide nanostructure or Titanium dioxide or Iron oxide nanostructure.

18. The composition or the device as claimed in claim 13, wherein the photolytic material is selected from a group consisting of Protoporphyrin (PPP), Porphyrin meso-tetrakis-[4-(carboxy methyleneoxy) phenyl] porphyrin (H2T4CPP), 6- hydroxy - l -methylene-3-oxo-3Hbenzopyrans, 6-methoxy - l -methylene-3-oxo- 3Hbenzopyrans, Nitrobenzyl, Aspartyl β-nitrobenzyl ester, chlorine peroxide molecule, 2-nitrobenzyl group, tetracationic monointercalant, cyclo-bisintercalant, 4,4-bipyridinium, oligonucleotides, oxazole yellow (YO), dimer of oxazole yellow (YOYO), dimer of thiazole orange (TOTO), cis- and trans- components.

19. The composition or the device as claimed in claim 13, wherein the linker is selected from a group consisting of l-ethyl-3-(3-dimethyIaminopropyl) (EDC), thio self assembly mono layer ( 1 ,8-octanedithiol); 1 ,4-bis(mercaptomethyl) benzene; 1 ,4- bis(merca tomethyl) cyclohexane; 1 ,4-bis (mercaptoacetamido) benzene; 1 ,4- bis(mercaptoacetamido) cyclohexane and sulfhydryl.

20. The composition as claimed claim 1 1 , wherein the biomolecule is selected from a group consisting of nucleic acid, DNA, RNA, SNP, cell marker proteins, cellular toxins, pclypeptide, peptide, amino acid, protein, fusion protein, antibody, antibody fragment, small molecules, lipids, and RNA or DNA virus.

21 . The composition as claimed in claim 1 1 , wherein the composition is selected from a group consisting of a solution, a suspension, a gel, a sol gel, a colloid, and a combination thereof.

22. The composition as claimed in claim 1 1 , wherein the composition further comprises a reporter molecule.

23. The device as claimed in claim 12, wherein the support is made up of a material selected from a group consisting of polymers, Acrylics, Cyclic Olefin Polymers,

Cyclic Olefin Co-polymers, Glass, Quartz, Silicon, Zinc, PolyCarbonates, Polypropylenes, Polyamides

24. The device as claimed in claim in claim 23, wherein the polymer is selected from a group consisting of polyvinylpyrrolidone, poly(ethylene oxide)-poly(propylene oxide)-poly(ethyleneo oxide) block co-polymer, a poly(ethylene oxide), poly(N- isopropyl acrylamide), polyefhyleneimine, polypyrolle, polyaniline. Poly 3,4- ethylenedioxy thiophene, polyacrylamide, polyvinyl alcohol or collagen.

25. A system comprising the composition as claimed in claim 1 1 .

26. The system as claimed in claim 25, wherein the system is selected from a group consisting of sensors, Medical Diagnostic and Therapeutic Tools, Hazardous

Material Detection Systems and Drug Screening system.