Direct detection of disease biomarkers in clinical specimens using cationic nanoparticle-based assays and versatile and green methods for synthesis of anisotropic silver nanostructures

Abstract

A gold nanoparticle-based assay for the detection of a target molecule, such as Hepatitis C Virus (HCV) RNA in serum samples, that uses positively charged gold nanoparticles (AuNPs) in solution based format. The assay has been tested on 74 serum clinical samples suspected of containing HCV RNA, with 48 and 38 positive and negative samples respectively. The developed assay has a specificity and sensitivity of 96.5% and 92.6% respectively. The results obtained were confirmed by Real-Time PCR, and a concordance of 100% for the negative samples and 89% for the positive samples has been obtained between the Real-Time PCR and the developed AuNPs based assay. Also, a purification method for the HCV RNA has been developed using HCV RNA specific probe conjugated to homemade silica nanoparticles. These silica nanoparticles have been synthesized by modified Stober method. This purification method enhanced the specificity of the developed AuNPs assay. The method can detect a target molecule, such as HCV RNA in serum, by employing modified silica nanoparticles to capture the target from a biological sample followed by detection of the captured target molecule using positively charged AuNPs. The assay is simple, cheap, sensitive and specific. Another aspect of the invention is anisotropic silver nanoparticles and methods of their use.

Claims

1. A method for detecting a nucleic acid in a sample comprising: contacting a sample suspected of containing a nucleic acid with silica nanoparticles that are bound to a capture probe which binds to the nucleic acid, thereby binding the nucleic acid to the capture probe on the silica nanoparticles, eluting the captured nucleic acid from the silica nanoparticles, contacting the eluted nucleic acid with positively charged silver nanoparticles, determining the aggregation of the silver nanoparticles after contacting them with the eluted nucleic acid, and selecting a sample containing the nucleic acid when the silver nanoparticles aggregate in comparison with a control sample that does not contain the nucleic acid to be detected; wherein nanoparticle aggregation is determined colorimetrically by a color change from yellow which denotes substantially nonaggregated silver nanoparticles, to colorless or to white which denotes aggregated silver nanoparticles.

2. The method of claim 1 that comprises detecting a nucleic acid that is single-stranded DNA.

3. The method of claim 1 comprising detecting a nucleic acid that is single-stranded RNA.

4. The method of claim 1, wherein the nanoparticles are spherical or spheroidal and have an average diameter of 12 to 40 nm.

5. The method of claim 1, wherein the nanoparticles are spherical or spheroidal and have an average diameter of 15-18 nm.

6. The method of claim 1, wherein the nanoparticles are not spherical or spheroidal.

7. The method of claim 1, wherein the silver nanoparticles are rod-shaped having any aspect ratio.

8. The method of claim 1, wherein the nanoparticles are star-shaped.

9. A method for capturing at least one biological material of interest comprising contacting a sample comprising said material with silica nanoparticles, which comprise a ligand for the biological material, for a time and under conditions sufficient for the material to bind to the silica nanoparticles and eluting or recovering the biological material from the silica nanoparticles; wherein said method further comprises contacting the eluted or recovered biological material with silver nanoparticles; wherein said at least one biological material is HCV RNA, wherein the ligand for the biological material comprises a nucleic acid complementary to the HCV RNA, and wherein said contacting comprises contacting the HCV RNA with silica nanoparticles conjugated to a nucleic acid complementary to HCV RNA, removing material that is not bound to the silica nanoparticles, eluting HCV RNA bound to the silica nanoparticles, contacting the eluted HCV RNA with cationic silver nanoparticles, and detecting HCV RNA when the cationic silver nanoparticles aggregate, wherein aggregation of cationic silver nanoparticles is determined colorimetrically by color change from yellow which denotes substantially non aggregated nanoparticles to colorless or to white which denotes aggregated nanoparticles.

10. A method for detecting a target molecule comprising: contacting a sample suspected of containing the target molecule with silica or magnetic nanoparticles that are bound to a capture probe or capture ligand for said target molecule, separating the silica or magnetic nanoparticles and any target molecule bound to the capture probe or capture ligand from other components of the sample, separating captured target molecule from the silica or magnetic nanoparticles, contacting said separated target molecule with positively charged silver nanoparticles that are not bound to capture probes or capture ligands, and selecting a sample containing the target molecule when the silver nanoparticles aggregate in comparison with a control sample that does not contain the target molecule to be detected; wherein the target molecule is a protein, wherein the silica or magnetic particles are bound to a capture ligand for the protein, and wherein nanoparticle aggregation is determined colorimetrically by a color change from yellow which denotes substantially nonaggregated silver nanoparticles, to colorless or to white which denotes aggregated silver nanoparticles.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1. Flow chart of one embodiment of a cationic AuNP-based assay. HCV virions are first lysed with RNA lysis buffer and proteins are digested by Proteinase K. HCV RNA present in the digested lysate is captured with silica nanoparticles conjugated to an oligonucleotide specific for the target HCV RNA. The captured target RNA is then washed and then eluted in a purified form. The purified RNA is added to AuNPs in a phosphate buffer. A color change from red to blue observed within about 2 minutes. This change in color occurs due to alignment of the positively charged AuNPs on the phosphate backbone of the HCV RNA. AuNPs in samples to which no RNA is added do not aggregate and remain red.

(2) FIG. 2. UV-Vis spectrum of the positive AuNPs. The spectrum shows a .sub.max at 531 nm characteristic of spherical AuNPs.

(3) FIG. 3. (FIG. 3A) SEM of the prepared AuNPs. (FIG. 3B) The particles have an average diameter of about 30 nm.

(4) FIG. 4. Analysis of clinical specimens using AuNP-based assay. In the absence of HCV RNA, the AuNPs are positively charged and are separated from each other by repulsion and the solution is red in color. On the other hand, in samples containing HCV RNA the AuNPs align on the phosphate backbone of the HCV RNA changing the color of these HCV RNA-positive samples from red to blue.

(5) FIG. 5. (FIG. 5A) SEM of the prepared silica nanoparticles. (FIG. 5B) Diameter calculation of the prepared silica nanoparticles.

(6) FIG. 6. (FIG. 6A) SEM of the prepared magnetic nanoparticles. (FIG. 6B) Diameter calculation of the magnetic nanoparticles. (FIG. 6C) FT-IR of magnetic nanoparticles functionalized with amino propyl tri-ethoxy silane (APES), The band at 583 cm.sup.1 corresponds to the FeO bond, the one at 1050 cm.sup.1 and 1380 cm.sup.1 corresponds to the vibrations of SiOCH.sub.2 structure and SiCH.sub.2 scissoring vibrations respectively, while the NH bending mode and stretching vibrations of the free amino groups are appeared at bands 1625 cm.sup.1 and 3436 cm.sup.1 respectively. Also, the anchored propyl group of the APTES is present at band 2923 cm.sup.1.

(7) FIG. 7 illustrates the mechanism of how cationic AuNPs aggregate along a strand of nucleic acid.

(8) FIG. 8 shows SEM photographs of 3d flower-like silver structures with hollow multi layers (3-15 layers), surface roughness (10-200 nm), external channels (1-4) surrounding the particles.

(9) FIG. 9. SEM of 3d flower-like silver structures with more multi layer (16-30 layers) of hollow, surface roughness (250-400 nm), with larger holes (50-200 nm), highly external channels (5-10) surrounding the particles.

(10) FIG. 10. 3D scaffold fibers like (FIG. 10B), flakes (FIG. 10C), and cluster (FIG. 10D) Silver structures before centrifugation (FIG. 10A). 3D scaffold like with hollow layer (10-50 nm in width and 100-500 nm length) and rough surface (150-500 nm). Silver flakes like 10-50 nm in width and 20-200 nm length.

(11) FIG. 11. Cumulative release of AgNPs over time from LAgNPs prepared with 1:1, 1:2, or 1:3 molar ratios of PC:Cho and 1 molar ratio of AgNPs. Diffusion of free AgNPs through cellulose membrane was used as a control.

(12) FIG. 12. Antimicrobial effects of nanoliposomes (FIG. 12C), free AgNPs (FIG. 12B), and LAgNPs (FIG. 12A) against four different bacterial strains. In all experiments, bacterial growth was determined by reading absorbance at 600 nm after 24 h of treatment. Nanoliposomes used were prepared using 1PC:3 Cho.

PART 1: DETAILED DESCRIPTION OF THE INVENTION

(13) Gold Nanoparticle-Based Assays.

(14) The inventors have developed for the first time an AuNP-based colorimetric method (solution phase) utilizing non-functionalized positively charged AuNPs for the direct detection of unamplified RNA and/or DNA molecules extracted from clinical specimens. This method can be applied to identify biomarkers of diseases such as nucleic acids and/or proteins from eukaryotic or prokaryotic sources particularly that of pathogens. In the examples shown below, Hepatitis C Virus (HCV) RNA was used as a model for testing the efficiency and specificity of the developed assay, which was used for the direct and specific detection of unamplified HCV RNA extracted from clinical samples.

(15) HCV is a major global health problem as it infects about 200 million individuals worldwide, with 3-4 million newly infected annually [7]. Chronic Hepatitis C develops in about 70-90% of cases and about 5-20% and 1-5% of chronically infected patients develop cirrhosis and hepatocellular carcinoma (HCC), respectively [8]. As illustrated in FIG. 1, this method can employ a selective extraction method that captures the nucleic acid of interest from clinical specimens and then colorimetrically identify the captured nucleic acid. This method is simple, rapid, highly sensitive, specific and cost-effective.

(16) HCV RNA was extracted using a commercially available viral RNA extraction kit (Promega). Extracted HCV RNA was then added to the positively-charged AuNPs. In case of positive samples, the positively-charged AuNPs bind to the negatively-charged phosphate groups on the HCV RNA target via electrostatic attraction between the negatively charged RNA phosphate backbone and the positively charged CTAB capping on AuNPs leading to Plasmon-Plasmon interaction and a change in the solution colour from red to blue [2, 13]. In the absence of HCV RNA (absence of any nucleic acid), the AuNPs are far from each other (repulsion between positively-charged particles) and thus the solution colour remains red (FIG. 4). Since the developed assay utilizes positively-charged AuNPs instead of negatively charged ones for HCV RNA detection, the test is more direct and simpler requiring minimal optimization. It is important to note that the red colour is stable even when left undisturbed for several days. Thus, the developed assay can simply and rapidly (about 5 min) determine any nucleic acid (RNA and/or DNA single or double stranded) in the solution. The reported sensitivity and specificity is 91%. and 94.8%, respectively.

(17) The presence of nucleic acid in the sample, leads to alignment of the positively charged AuNPs on the phosphate backbone of the nucleic acid, the presence of phosphate buffer in the assay increases the aggregation capability of the AuNPs (for only the positive samples) by binding of the phosphate ions to the aligned AuNPs and then another row of AuNPs attached to the other part of the phosphate ions which will be aligned on another phosphate backbone of another RNA molecule (FIG. 7). So, aggregation occurs by first alignment of AuNPs on phosphate backbone of one nucleic acid, which then binds to phosphate ions and the latter binds to another AuNPs which are aligned on another nucleic acid phosphate backbone. Therefore, at least 5 layers are formed in the positive samples and leads to AuNPs aggregation, the layers are: 1stRNA/AuNPs/Phosphate ions/AuNPs/2ndRNA. These layers will result in forming a network of nucleic acid/AuNPs which lead to aggregation of gold nanoparticles and change of colour from red to blue.

(18) To increase specificity and sensitivity of the assay, HCV RNA specific probe conjugated to silica and/or magnetic nanoparticles have been used for viral RNA capture after lysis the virion and precipitation of the proteins in the sample so a simple, rapid, specific and direct magnetic/silica-probe based extraction method has been developed and when coupled with the AuNP based colorimetric assay a sensitivity and specificity of 92.6% and 96.5%, respectively, was obtained.

(19) The invention is not limited to a particular method for synthesizing cationic (positively charged) gold nanoparticles (AuNPs) or silver nanoparticles (AgNPs). Various methods may be used. General synthesis methods for producing positively charged gold nanoparticles are based mainly on the reduction of hydrogen tetrachloroaurate trihydrate (HAuCl.sub.4) using sodium borohydride as a reducing agent, in the presence of the capping agent. Changing concentration of the different reagents, reaction time and pH will determine the final size and shape of the prepared nanoparticles. The common used capping agents are:

(20) (i) cetyl trimethyl ammonium bromide (CTAB), see Narayanan R, Lipert R. J, Porter M D. Cetyltrimethylammonium bromide-modified spherical and cube-like gold nanoparticles as extrinsic Raman labels in surface-enhanced Raman spectroscopy based heterogeneous immunoassays. Analytical chemistry. 2008, 80(6): 2265-71; and Wenlong Cheng, Shaojun Dong, Erkang Wang. Synthesis and self assembly of cetyltrimethylammonium bromide capped gold nanoparticles. Langmuir. 2003, 19(22): 9434-39 (both of which are incorporated by reference);

(21) (ii) cysteamine, see Kim J W, Kim J H, Chung S J, Chung B H. An operationally simple colorimetric assay of hyaluronidase activity using cationic gold nanoparticles. Analyst. 2009 July; 134(7):1291-3; or T. Niidome et al. Preparation of primary amine-modified gold nanoparticles and their transfection ability into cultivated cells. Chem. Commun. 2004, (17):1978-1979 (both of which are incorporated by reference); and

(22) (iii) lysine, see P R. Selvakannan et al. Capping of gold nanoparticles by the amino acid lysine renders them water dispersible. Langmuir. 2003, 19(8): 3345-49 (which is incorporated by reference).

(23) Methods used for synthesis of positively charged silver nanoparticles (AgNPs), are similar to those used for the gold nanoparticles [9], and based on the reduction of silver salt in the presence of capping agents as CTAB [10, 11], polyethylimine (PEI) [12] and p-benzoquinone [13]. Silver nanoparticles based assays are based on change of color of the silver nanoparticles solution from yellow to colorless and/or formation of white precipitate based on the degree of aggregation.

(24) The present invention is described below based on some specific examples which pertain to some specific embodiments of the invention. However, the invention is not limited to what is described in these examples.

EXAMPLES

Example 1: Detection of HCV RNA in Serum Samples

(25) Synthesis of Positively-Charged AuNPs.

(26) Positively charged spherical particles were synthesized as previously described in [14]. Briefly, the seed solution was prepared by reducing HAuCl.sub.4 (2.5 ml of 0.001 M HAuCl.sub.4), in presence of CTAB (7.5 ml of 0.2 M), with ice-cold NaBH.sub.4 (600 L; 0.01 M). The vials were then shaken vigorously (about 2 min) to produce brown seed suspensions. The seed (80 l) was then added to the centre of a solution containing HAuCl.sub.4 (50 ml of 0.001 M HAuCl.sub.4), CTAB/BDAC mixture (50 mL containing 0.2 M of CTAB plus 0.25M of BDAC), AgNO.sub.3 (1.5 mL of 0.004 M AgNO.sub.3) and ascorbic acid (700 l of 0.0788 M). The mixture was left undisturbed for about 24 h.

(27) Characterization of Synthesized AuNPs.

(28) The absorbance spectrum and concentration of the positively charged particles were determined using UV spectrophotometer (Jenway 6800) as previously reported in [15]. The shape and size of the produced positively charged particles were analyzed using field emission scanning electron microscopy (SEM; Model: Leo Supra 55; US). For the SEM analysis, 5 l of the synthesized AuNPs were placed on silicon wafer and allowed to air dry before examination. UV-Vis spectrum was performed for the prepared AuNPs. The spectrum shows a .sub.max at 531 nm characteristic of spherical AuNPs (FIG. 2) [14]. Scanning Electron Microscope (SEM) image (FIG. 3A) was analyzed by (Image J 1.4 software Wayne Rasband, National Institutes of Health, USA. http://_rsb.info.nih.gov/ij/java 1.6.0_05). The particles had an average diameter of about 30 nm and spherical in shape (FIG. 3B).

(29) Isolation of Nucleic Acid from Serum

(30) Serum Sample Collection.

(31) Serum samples were collected from healthy volunteers (n=38) and from chronic HCV patients (n=48). Rapid HCV test was performed on all the samples. All positive samples had elevated ALT and AST levels. All samples were negative for hepatitis B surface antigen and hepatitis B antibody.

(32) Extraction of HCV RNA.

(33) Extraction of HCV RNA from serum samples was performed using SV total RNA isolation System (Promega; Cat. No. Z3100) according to the modified manufacturer's protocol for HCV RNA isolation [16].

(34) Real-Time RT-PCR.

(35) Real-time RT-PCR was performed using AgPath ID One Step RT-PCR kit (cat #AM1005; Ambion) [17] according to manufacturer's protocol. To 16.5 l master mix, 8.5 l of the extracted HCV RNA was added and amplification was performed using Stratagene (Mx3005P) under the following cycling conditions: 1 cycle of 45 C. for 10 min, 1 cycle of 95 C. for 10 min followed by 45 cycles of 95 C. for 15 s and 60 C. for 45 s.

(36) Colorimetric AuNP-Based Assay.

(37) To 5 L of the extracted HCV RNA, 5 L of 2M Phosphate buffer was added followed by 30 L of the positively charged AuNPs. The sample was mixed by pipetting and the color of the solution was observed within 5 minutes.

Example 2: Silica Nanoparticle Capturing Method

(38) Colloidal Silica Nanoparticles: Synthesis and Functionalization.

(39) HCV RNA was extracted using colloidal silica nanoparticles conjugated to an oligonucleotide specific to HCV RNA. Initially, 200 nm colloidal silica nanoparticles were synthesized with a modified stober method (modification was done in our lab). Briefly, in a beaker mix absolute ethanol, deionized water, concentrated ammonia and tetraethyl orthosilicate (TEOS), and stir at room temperature for about 1 hour. Then, the formed colloidal solution was centrifuged at 4000 rpm for 10 minutes, and the supernatant was discarded and the pellet was washed with absolute ethanol. This washing step was repeated for about 4 times or until no ammonia odor in the solution. The pellet was then dispersed in absolute ethanol and sonicated for about 5 minutes to remove any aggregates. The produced silica nanoparticles were examined using scanning electron microscope (SEM), to get the morphology and the diameter of the prepared silica nanoparticles (FIG. 5).

(40) The prepared silica nanoparticles were functionalized with amino propyl trimethoxy silane (APMS) to introduce amino groups on the surface of the silica nanoparticles. Briefly, 1 ml of APMS was added to 20 ml of the prepared colloidal silica nanoparticles and stirred at room temperature for at least 2 hours, then the solution was centrifuged at 4,000 rpm for 10 minutes, and the supernatant was discarded and the pellet resuspended in phosphate buffer saline (PBS 1). The number of silica nanoparticles per ml was calculated as previously described [18]. Briefly, one ml of the prepared silica colloidal solution was taken centrifuged, and the supernatant was discarded, then the pellet was dried till complete dryness. The dried pellet was then weighted in milligrams and from the volume taken (1 ml) and the weight obtained, concentration of the colloidal solution has been calculated which is 12 mg/ml. From FIG. 6 the diameter of the silica nanoparticles was measured to be about 150 nm. The weight of one particle equals volume of the particle*specific gravity (2.3), the volume equals 4/3 r3, and the particles count equals concentration (12 mg/ml)/weight of one particle equals 2.95125E+12 silica nanoparticles/ml.

(41) Synthesis of Silica Probe.

(42) A heterobifunctional cross linker (3-maleimidobenzoic acid N-hydroxyl succinimide, MBS) was used to prepare an HCV specific probe conjugated to silica nanoparticles. This linker has NHS ester at one end which reacts with primary amine groups to form stable amide bond; the other end has maleimide group which reacts with sulfhydryl groups. The cross linker binds to the amine functionalized silica nanoparticles through the NHS ester and to a thiolated HCV specific probe through the maleimide group. The thiol labeled probe was prepared as previously described [19, 20]. 10 mg of MBS dissolved in 1 mL dimethyl formamide plus 2.5 ml PBS and 2.5 ml of amino functionalized silica nanoparticles and mix at room temperature for at least 2 hours, and then purified by centrifugation, the thiol modified probe was added to the MBS conjugated silica nanoparticles and incubate at room temperature for at least 2 hours. The number of probes per one silica nanoparticle was calculated by first multiplying the number of moles of the probe by Avogadro's number, and then dividing the number of probes calculated by the silica nanoparticles count, and it was about 500 probes per one silica nanoparticle.

(43) Extraction of HCV RNA from Clinical Samples Using the Prepared Silica Probes.

(44) To 200 11.1 of patient sera, 200 l of lysis buffer (Promega SV viral RNA) was added. After mixing by inversion, 50 l Proteinase K was added and left to incubate for 10 min. The mixture was heated to 95 C. in a heat block for 2 min then 50 l silica-probes was added and the reaction mixed for 1 hr. The mixture was centrifuged at 3000 RPM for 3 min and the pellet was washed twice with nuclease-free water. The HCV RNA was then eluted by heating at 95 C. for 5 min. The mixture was centrifuged and the supernatant contained the eluted RNA was separated. The extracted RNA was tested using both Real-time RT-PCR and the developed colorimetric AuNP-based assay.

(45) Comparison Between Colorimetric AuNP-Based Assay and Real-Time RT-PCR.

(46) HCV RNA extracted by the Promega kit or by the silica probe developed by the inventors was detected and quantified using Real-Time RT-PCR as described above. The assay developed by the inventors using positively charged AuNPs was performed on the samples extracted using the silica probe to detect HCV RNA. The color of the AuNPs colloidal solution of the negative samples remained red in color which indicates no nucleic acid was present in the sample.

(47) On the other hand, the presence of HCV RNA in the positive samples lead to aggregation of the AuNPs and the color changed from red to blue (FIG. 4), the intensity of the blue color in positive samples was compatible with the viral load as quantified by Real-Time PCR. Of the 68 HCV positive samples, 63 samples gave blue color which indicates the presence of the HCV RNA, while no change in color occurred in 5 samples (False negative).

(48) On the other hand, 56 out of 58 negative samples gave red color which indicates the absence of HCV RNA in addition to any other nucleic acid (high purity of the sample), while one sample only gave blue color due to AuNPs aggregation (False Positive). These results show that the cationic AuNP based assay has specificity of 96.5%, and a sensitivity of 92.6%.

Example 3: Magnetic Nanoparticle Capturing Method

(49) Iron Oxide Magnetic Nanoparticles: Synthesis and Functionalization.

(50) HCV RNA was extracted using homemade magnetic nanoparticles conjugated to an oligonucleotide specific to HCV RNA. First, 90 nm magnetic nanoparticles were synthesized as described elsewhere [21]. Typically iron (II) chloride and iron (III) chloride (1:2) were dissolved in nanopure water at the concentration of 0.25 M iron ions and chemically precipitated at room temperature (25 C.) by adding 1 M NaOH at a constant of pH 10. The precipitates were heated at 80 C. for 35 min under continuous mixing and washed four times in water and several times in ethanol. During washing, the magnetic nanoparticles were separated from the supernatant using a magnet, and the particles were finally dried in a vacuum oven at 70 C.

(51) Amino functionalization of the prepared magnetic nanoparticles was done by amino propyl Trimethoxy silane (APMS) as described elsewhere [22]. Briefly, magnetic nanoparticles (1 g) were washed with 99.5% methanol and twice with Nanopure water and soaked in 10 mL of 3 mM APTMS solution in a toluene/methanol (1:1 v/v) mix. The suspension was then transferred into a three-necked flask with a water-cooled condenser and temperature controller with a nitrogen gas flow at 80 C. for 20 h under vigorous stirring. Silanization was found to occur at the surfaces of the particles bearing hydroxyl groups, which in the presence of an organic solvent results in the formation of an APTMS coating with a large density of amines. The particles were recovered by applying an external magnetic field after the silanization process and washed three times with methanol and dried at 50 C. in a vacuum oven.

(52) Characterization of Magnetic Nanoparticles.

(53) The prepared magnetic nanoparticles were characterized by SEM for size and particles distribution determination. Moreover, Fourier transform infrared spectroscopy (FT-IR) was used to record the IR spectra of the samples using potassium bromide (KBr) pellet technique.

(54) Conjugation of HCV Specific Probe to the Amino Functionalized Magnetic Nanoparticles.

(55) To prepare the magnetic nanoparticles conjugated to HCV RNA specific probe, heterobifunctional cross linker (3-maleimidobenzoic acid N-hydroxyl succinimide, MBS) was used that has NHS ester at one end that reacts with primary amine groups forming stable amide bond, and the other end has maleimide group that reacts with sulfhydryl groups forming stable thioether linkage. Functionalization procedures were done as following: Firstly, the disulfide labeled probe was prepared as previously described [19, 20]. Briefly, disulfide cleavage of the probe was done by lyophilization of 10 nmol of the probe and then resuspended in 100 ul of 0.1 M dithiothrietol (DTT) prepared in disulfide cleavage buffer (170 mM phosphate buffer, pH=8). The solution was wrapped in foil and let to stand at room temperature for 2-3 h with occasional vortexing. Desalting of the freshly cleaved probe was done using Nap-5 column (illustra NAP-5 (GE Healthcare) according to the manufacturer's instructions. UV-visible spectrophotometer was used to determine the purified probe concentration. Secondly, the amine functionalized magnetic nanoparticles were washed with Dimethyl sulfoxide (DMSO) for 2 times, the wash discarded, then MBS cross linker dissolved in DMSO was added to the washed magnetic nanoparticles The mixture was allowed to mix on a roller shaker for about 1 hour at room temperature. Then, the nanoparticles were washed with DMSO twice followed by coupling buffer (100 mM phosphate buffer and 0.2 M sodium chloride, pH=7) twice. Then, the particles were resuspended again in coupling buffer and the cleaved probe were added to the suspended particles and allowed to react on a roller shaker overnight. Finally, the supernatant was removed and the magnetic nanoparticles functionalized with HCV RNA specific probe were resuspended in storage buffer (10 mM phosphate buffer, 0.1 M sodium chloride, pH=7.4).

(56) Extraction of HCV RNA from Clinical Samples Using the Prepared Probes.

(57) To employ specificity and selectivity of the developed AuNP assay for a specific viral RNA/DNA, HCV RNA specific probe conjugated to magnetic nanoparticles was used for HCV RNA capture after virion lysis and digestion of the proteins in patient sera. In a 1.5 ml microcentrifuge tube, 100 ul of the magnetic nanoparticles conjugated to HCV specific probe was taken and washed twice with the assay buffer (10 mM phosphate buffer, 150 mM sodium chloride, pH=7.4) and then the modified magnetic nanoparticles were resuspended in 50 ul assay buffer.

(58) In another 1.5 ml microcentrifuge tube 200 l of patient sera was added to 200 l of lysis buffer to break down the viral envelope. After mixing by inversion, 50 l Proteinase K was added to digest the serum proteins and left to incubate for 10 min. Then, the mixture was centrifuged for 10 minutes at 14,000 rpm and the supernatant was taken and mixed with 300 ul of iso-propanol. Then, the resuspended magnetic nanoparticles were added to the previous mixture and heated at 90 C. for 2 minutes to denaturate the target RNA. The mixture was shaken at a temperature 15 C. below the melting point of the conjugated probe for 45 minutes. Then, the tubes were placed on magnet until all solutions were clear and the supernatant were removed. Then, the particles were washed twice with washing buffer (60 mM potassium acetate, 10 mM Tris-HCl, 60% ethanol, pH=7.5). Supernatant was removed between each wash with the help of magnet. Elution was done by adding 50 l DEPECWater, and heated at 95 C. for 2 minutes. The tubes were placed on magnet until all solutions were clear and the eluted HCV RNA was transferred to new RNase free tube. After HCV RNA extraction the colorimetric gold nanoparticles based assay was performed as described before.

(59) HCV RNA Capturing.

(60) One hundred twenty six samples were used in this study: 68 samples from HCV positive subjects and 58 samples from healthy individuals. Each sample was divided and each part was subjected to extraction by one of two different methods: (i) by use of an SV total RNA isolation kit or (ii) by use of the developed silica and/or magnetic probe. The presence of the HCV RNA in the positive samples and the absence of HCV RNA in the negative samples extracted by the silica probe were confirmed with Real-Time PCR and compared with the samples extracted by the promega kit. The concordance between RNA extraction by the promega kit and the RNA extraction by the silica and/or magnetic probe was 100%, (which means that the developed silica and/or magnetic probe could be used alone for HCV RNA extraction without the need of any other commercial extraction kit. Moreover, the same principle can be used in extraction and purification of any other nucleic acid and/or protein by simply replace the HCV RNA specific probe with the other target specific molecule (e.g. antibodies, lectins, probes . . . etc), therefore the developed HCV RNA extraction method by the silica probe could be expanded to be used in many other targets. The main aim for capturing the HCV RNA capturing is to increase the purity of the sample from any other nucleic acids (DNA or RNA) that may interfere with the assay results and thus allowing an increase in assay specificity.

(61) Other Applications.

(62) The inventors have developed a novel colorimetric solution-phase cationic AuNP-based assay for the direct detection of unamplified RNA/DNA. This method has been exemplified using HCV RNA extracted from clinical specimens as a model nucleic acid. The developed assay is not complex because it simply requires adding cationic AuNPs solution to the target molecule (e.g., extracted RNA in presence of phosphate buffer, rapid (about 5 min), sensitive, cost-effective, and can be easily automated. Conveniently, the developed assay can be used as a platform for the detection of any RNA and/or DNA in solution form. In other words, if a specific nucleic acid extraction method is available for any nucleic acid to be determined; this assay provides an easy, simple and rapid way for its analysis.

(63) This method provides a foundation for development of other silica-probe based extraction methods for extraction of specific kinds of nucleic acids, such as HCV RNA. This assay can be practiced quantitatively since the intensity of the blue colour produced by aggregation of gold and/or silver nanoparticles reflects the number of RNA/DNA molecules present in the extracted sample and corresponds to factors such as bacterial or viral load. Based on the disclosure above one of skill in the art could determine the qualitative or quantitative detection limit of an assay for a particular kind of nucleic acid, its linear range, accuracy and precision.

REFERENCES FOR PART 1

(64) 1. Radwan, S. H. and H. M. Azzazy, Gold nanoparticles for molecular diagnostics. Expert Rev Mol Diagn, 2009. 9(5): 511-24. 2. Cao R, et al., Naked-eye sensitive detection of nuclease activity using positively-charged gold nanoparticles as colorimetric probes. Chem Commun., 2011. 47(45): 12301-12303. 3. Kim J W, et al., An operationally simple colorimetric assay of hyaluronidase activity using cationic gold nanoparticles. Analyst, 2009. 134(7): 1291-1293. 4. Ma Z, et al., Optical DNA detection based on gold nanorods aggregation. Anal chem Acta, 2010. 673(2): 179-184. 5. Sun, Y., et al., Microarray gene expression analysis free of reverse transcription and dye labeling. Anal Biochem, 2005. 345(2): 312-9. 6. Hsiao C R and C. C H, Characterization of DNA chips by nanogold staining. Anal Chem, 2009. 389(2): 118-123. 7. WHO http://www.who.int/vaccine_research/diseases/hepatitis_c/en/ [last accessed on Jul. 22, 2010]. 8. Gourley, P. L., Brief overview of BioMicroNano technologies. Biotechnol Prog., 2005. 21 (1): 2-10. 9. Tolaymat, T. M., et al., An evidence-based environmental perspective of manufactured silver nanoparticle in syntheses and applications: A systematic review and critical appraisal of peer-reviewed scientific papers. science of the total environment, 2009. 408: 999-1006. 10. Sui, Z., et al., Capping effect of CTAB on positively charged Ag nanoparticles. Physica E.: Low-dimensional systems and nanostructures, 2006. 33(2): 308-314. 11. Khan, Z., et al., Preparation and characterization of silver nanoparticles by chemical reduction method. Colloids and Surfaces B: Biointerfaces, 2011. 82: 513-517. 12. Siliu Tan, et al., Synthesis of positively charged silver nanoparticles via photoreduction of AgNO3 in branched Polyethyleneimine/HEPES solutions. langmuir, 2007. 23(19): 9836-9843. 13. Kima, J., S. W. Kang, and Y. S. Kang, Partially positively charged silver nanoparticles prepared by p-benzoquinone. Colloids and SurfacesA: Physicochem. Eng. Aspects, 2008. 320: 189-192. 14. Huang X: Gold Nanoparticles Used in Cancer Cell Diagnostics, Selective Photothermal Therapy and Catalysis of NADH Oxidation Reaction. Laser Dynamic Laboratory, School of Chemistry and Biochemistry Doctor of philosophy, 234 pages (2006). 15. Jain, P. K., et al., Calculated Absorption and Scattering Properties of Gold Nanoparticles of Different Size, Shape, and Composition: Applications in Biological Imaging and Biomedicine. J. Phys. Chem. B, 2006. 110(14): 7238-7248. 16. Link, S. and M. A. El-Sayed, Spectral Properties and Relaxation Dynamics of Surface Plasmon Electronic Oscillations in Gold and Silver Nanodots and Nanorods. J. Phys. Chem. B, 1999. 103(40): 8410-8426. 17. Wagner, V. et al., The emerging nanomedicine landscape. Nat Biotechnol, 2006. 24(10): 1211-7. 18. Nakamura, M., M. Shono, and K. Ishimura, Synthesis, characterization, and biological applications of multifluorescent silica nanoparticles. Anal Chem, 2007. 79(17): 6507-14. 19. Rosi, N. L., et al., Oligonucleotide-modified gold nanoparticles for intracellular gene regulation. Science, 2006. 312: 1027-1030. 20. Haley D Hill and C. A. Mirkin, The bio-barcode assay for the detection of protein and nucleic acid targets using DTT-induced ligand exchange. Nature Protocols 2006. 1: 324-335. 21. Kouassi, G. K., J. Irudayaraj, and G. McCarty, Activity of glucose oxidase functionalized onto magnetic nanoparticles. Biomagn. Res. Technol., 2005. 3(1): 1. 22. Kouassi, G. K. and J. Irudayaraj, Magnetic and gold-coated magnetic nanoparticles as a DNA sensor. Anal. Chem, 2006. 78(10): 3234-41.

PART 2: DETAILED DESCRIPTION OF THE INVENTION

(65) Anisotropic Silver Nanoparticles.

(66) The synthesis of anisotropic silver nanoparticles is a time-consuming process and involves the use of expensive toxic chemicals and specialized laboratory equipment. The presence of toxic chemicals in the prepared anisotropic silver nanostructures hindered their medical application. The inventors have developed a fast and inexpensive method for the synthesis of three dimensional hollow flower-like silver nanostructures without the use of toxic chemicals. In this method, silver nitrate was reduced using dextrose in presence of trisodium citrate as a capping agent. Sodium hydroxide was added to enhance reduction efficacy of dextrose and reduce time of synthesis. The effects of all four agents on the shape and size of silver nanostructures were investigated. Robust hollow flower-like silver nanostructures were successfully synthesized and ranged in size from 0.2 m to 5.0 m with surface area between 25-240 m.sup.2/g. Changing the concentration of silver nitrate, dextrose, sodium hydroxide, and trisodium citrate affected the size and shape of the synthesized structures, while changing temperature had no effect. The disclosed method is simple, safe, and allows controlled synthesis of anisotropic silver nanostructures, which may represent promising tools as effective antimicrobial agents and for in vitro diagnostics. The synthesized hollow nanostructures may be used for enhanced drug encapsulation and sustained release.

(67) Particular embodiments of the invention are described below: Methods that control of size and morphology of synthesized nanostructures that do not require the use of particular polymers, surfactants and/or special laboratory equipment. These methods are used for synthesizing novel silver nanostructures having the following characteristics:

(68) sizes ranging from 0.17 to 7 M having distributed pores ranging in size from 10 to 20 nm and that have a 3 dimensional (3d) flower-like structure with multilayer hollows, rough surfaces, external channels and distributed interior hollows;

(69) unique flower like silver structures with multi layer of hollows in the range of 3-15 layers, surface roughness ranged from 10-200 nm, more rough surface, with larger holes (10-20 nm in width and 200-300 nm in length), highly external channels in the range of 1-4 channels surrounding silver structures with size ranging between 0.2-3.0 M. Likewise 3d flower-like silver structures with size in the range of 0.25-2.5 M with more multi layers (16-30 layers), surface roughness ranged from 250-400 nm, with larger holes ranged from 50-200 nm, highly external channels ranged from 5-10 channels surrounding silver particles.

(70) unique 3d shell-like silver structures with little pores ranged between 5-30 nm, rough surface (180-300 nm), highly external arms ranged from 3-9 arms surrounding particles, having size ranging between 0.15-1 M.

(71) unique scaffold like silver structures having size ranging between 2-7 M with highly inter connected pores ranging between 50-100 nm width and 100-500 nm length;

(72) unique roll fiber twin like silver structures having size ranging between 0.15-1 M with multi hard external arms on the surface complete shelled the particles;

(73) unique 3d pores spherical silver structures have size in the range 0.6-5 M of with well distributed external pores on the surface ranges between 50 nm-300 nm;

(74) unique 3d pores spherical silver structures spongy like with size in the range of 0.3-2 M with well interconnected pores in the range of 20-100 nm;

(75) flower like with multi external interacted layers of hollows, branched rough edges silver nanostructures with size in the range of 0.3-1.5 M with well controlled size and dispersion;

(76) flower-like silver structures with multi layer of paper, hollows like cores, soft surface silver nanostructures with size in the range of 0.2-1 M with well controlled size and dispersion;

(77) trees with multi branched edges like arms like silver nanostructures with size in the range of 2-6 M with well controlled size and dispersion;

(78) dendrimer silver nanostructures with size in the range of 0.7-2 M with well controlled size and dispersion;

(79) flower silver structures with more internal hollows, rough surface silver nanostructures with size in the range of 0.24-1.5 M with well controlled size and dispersion;

(80) cubes silver structures with size in the range of 0.1-1 M with well controlled walls and edges;

(81) pyramidal silver structures with size in the range of 0.05-2 M with well controlled walls, edges and soft surface;

(82) bibode silver structures with size in the range of 50-200 nm with soft surface;

(83) triangular silver structures with size in the range of 0.1-1 M;

(84) octahedral multi layer silver structures with size in the range of 100-400 nm;

(85) octahedral multi layer, with pores on the surface silver structures with size in the range of 100-300 nm;

(86) ribbed like silver structures with size in the range of 0.3-1 M; multi ribbed with cubes decorated on the surface silver structures with size in the range of 100-600 nm;

(87) octahedral with cube decorated on the surface silver structures with size in the range of 0.2-1 M;

(88) silver stars with size in the range of 0.1-4 M and with soft branched arms and hole as core like;

(89) silver stars with size in the range of 0.15-3 M and with soft and rough, branched, more layer of arms and hole as core like;

(90) silver flower structures with size in the range of 100-400 nm with soft, more layers of arms;

(91) silver myriad dendrimer structures with size in the range of 0.5-2 M, with highly branched arms and core like;

(92) silver butterfly structures with size in the range of 0.6-1.5 M and with highly branched arms like wings;

(93) silver stars, flower structures with size in the range of 0.3-2 M and by incorporation of polymers with TSC and using UV irradiation; and

(94) silver stars, flower structures with size in the range of 0.2-1 M and by incorporation of polymers with TSC and without using of UV irradiation.

(95) Synthesis of Anisotropic Silver Structures.

(96) The anisotropic silver structures silver nanostructures, and microstructures with control in size, shape were synthesized by chemical reduction of silver nitrate with dextrose in presence of trisodium citrate and sodium hydroxide (NaOH). Dextrose acts as reducing agent, as capping agents TSC act only as capping material, NaOH acted enhance reduction efficacy of dextrose and as shape control.

(97) Likewise, cubes, pyramidal, triangular, silver nanostructures, macrostructures, are prepared by incorporation of ascorbic acid with TSC, dextrose and NaOH. By the same way dextrose act as reducing agent, as capping agents TSC act only as capping material, NaOH acted enhance reduction efficacy of dextrose, Ascorbic acid and as shape control. The particles morphology, size were controlled by reaction condition include amount of TSC, AgNO.sub.3, dextrose, ascorbic acid, and NaOH as will be demonstrated and discussed below. The methods demonstrated herein provide nanostructures, macrostructures with high uniformity in size, controllable size, morphology, large quantities, reproducibly and good solubility in various solvents. In addition to such method are fast, echo-friendly, and inexpensive, with lack of specially laboratory equipments and laboratory skills.

(98) A round bottom glass flask (100 mL) were cleaned in aqua regia (3 parts HCl, 1 part HNO.sub.3) and rinsed with DDI water and dried in the oven at 60 C. All the other glasses were should be cleaned by the same way.

(99) The formulas included silver nitrate AgNO.sub.3 (silver source), trisodium citrate salts, anhydrous (TSC) (shape control reagents), dextrose (reducing agents, shape controller), L-Ascorbic acid (ascorbic acid) as reducing agents, Sodium hydroxide pellets, 97% (NaOH) and DDI water as solvents all above reagents were provided from Sigma Aldrich and water provided from mille pore (Millipore Corporation, Billerica, Mass.) with a resistivity of 18 Mcm in our lab.

(100) A solution of AgNO.sub.3 were prepared by dissolved appropriate amount of AgNO.sub.3 in DDI water with final concentrations of 0.001-2.0 M and 0.001-0.1 mM, and vortex for 1.5 min to complete dissolution and covered with aluminum fuel to protect from light. A solution of TSC was prepared by dissolving appropriate amount of TSC in DDI water with final concentrations of 0.001-2.0M and vortex for 1.5 minute to complete dissolution. A solution of Ascorbic Acid were prepared by dissolved appropriate amount of Ascorbic Acid in DDI water with final concentrations of 0.3-3.0 M and vortex for 2 min to complete dissolution and covered with aluminum fuel to protect from light. A solution of dextrose were prepared by dissolved appropriate amount of dextrose in DDI water with final concentrations of 0.001-2.0 M and vortex for 2 min to complete dissolution and cover with aluminum fuel to protect from light. A solution of NaOH was prepared by dissolved appropriate amount of NaOH in DDI water with final concentrations of 0.001-5.0 M and vortex for 2 min to complete dissolution. All the above reagents should be stored in sterilized Falcon tubes.

(101) Synthesis of Nanoparticles Having 3d Flower-Like Morphology.

(102) The 3d flower like morphology with distributed interior hollow structures; branched edges are synthesized by the chemical reduction of silver nitrate in aqueous phase. Briefly 0.001-2.0 M AgNO.sub.3, 0.001-2.0M TSC solution, 0.3-3.0M of dextrose added to 15 mL DDI, then added various concentration of NaOH 0.001-5.0 M, stirred at room temperatures the color change immediately to deep gray or green, deep yellow depended on the reaction condition after the color changed, the solution is stirred for an additional 5 minutes, and stirrer turn off. The particles were collected by centrifugation at 1400 rpm for 10 minute, the supernatant was discarded and precipitate re-suspended in DDI water; the process was repeated three times to remove excess dextrose.

Example 1

(103) The 3d flower-like silver structures with multi layer of hollow, rough surface, external channels surrounded particles were synthesized according to the above condition but using 0.2 mL of AgNO.sub.3, 0.4 mL of TSC, 0.4 mL of dextrose added to 15 mL of DDI water; stirring at room temperature the color turned to deep yellow immediately after addition of 100 L of NaOH then the solution is stirred for an additional 5 min, stirrer turned off and samples collected by centrifugation as mentioned above (FIG. 8).

Example 2

(104) The 3d flower-like silver structures with more multilayer of hollow, more rough surface, with larger hollows, highly external channels surrounded particles were synthesized according above condition but using 0.2 mL of AgNO.sub.3, 0.4 mL of TSC, 0.4 mL of dextrose added to 15 mL of DDI water, stirring at room temperatures the color turn to gray immediately after addition of 250 L of NaOH then the solution is stirred for an additional 5 minutes, stirrer turned off and samples collected by centrifugation as mentioned above (FIG. 9).

Example 3

(105) The 3D scaffold fibers like, flakes, and cluster silver structures little hollow pores, more rough surface, highly external arms surrounded particles were synthesized according above condition but using 1 mL of AgNO.sub.3, 1 mL of TSC, 1 mL of dextrose added to 15 mL of DDI water, stirring at room temperatures the color turn to green immediately after addition of 50 L of NaOH then the solution is stirred for an additional 5 min, stirrer turned off and samples collected by centrifugation 3 times at 8,000, 12,000, and 14,000 rpm, respectively.

Example 4

(106) The 3d scaffold-like silver structures with little pores hollow, more rough surface, were synthesized according above condition but using 0.3 mL of AgNO.sub.3, 0.6 mL of TSC, 0.6 mL of dextrose added to 15 mL of DDI water, stirring at room temperatures the color turn to deep green immediately after addition of 100 L of NaOH then the solution is stirred for an additional 5 minutes, stirrer turned off and samples collected by centrifugation as mentioned above.

Example 5

(107) The 3d roll fiber twin-like silver structures with little pores hollows, more rough surface, were synthesized according above condition but using 0.3 mL of AgNO.sub.3, 0.6 mL of TSC, 0.6 mL of dextrose added to 15 mL of DDI water, stirring at room temperatures the color turn to deep green immediately after addition of 250 L of NaOH then the solution is stirred for an additional 5 min, stirrer turned off and samples collected by centrifugation as mentioned above.

Example 6

(108) The porous spheroid silver structures with little pores hollow, more rough surface, were synthesized according above condition but using 1 mL of AgNO.sub.3, 1 mL of TSC, 1 mL of dextrose added to 15 mL of DDI water, stirring at room temperatures the color turn to deep green immediately after addition of 200 L of NaOH then the solution is stirred for an additional 5 minutes, stirrer turned off and samples collected by centrifugation as mentioned above.

Example 7

(109) The porous spherical sponge-like silver structures with high inter connected pores, more rough surface, were synthesized according above condition but using 1 mL of AgNO.sub.3, 1 mL of TSC, 1 mL of dextrose added to 15 mL of DDI water, stirring at room temperatures the color turn to deep green immediately after addition of 100 L of NaOH then the solution is stirred for an additional 5 minutes, stirrer turned off and samples collected by centrifugation as mentioned above.

Example 8

(110) The 3d flower-like silver structures with multi external interacted layers of hollow, branched rough edges were synthesized according to above condition but using 1 mL of AgNO.sub.3, 1 mL of TSC, 1 mL of dextrose added to 10 mL of DDI water, added 50 L of NaOH stirring with heating up to 100 C. the color turn to yellow to deep brown immediately after 5 min then the solution is stirred till to cooling to room temperatures and stirrer turned off and samples collected by centrifugation as mentioned above.

Example 9

(111) The flower like silver structures with multi layer of paper, hollow-like cores, soft surface, were synthesized according to above condition but using 2.0 mL of AgNO.sub.3, 2 mL of dextrose, stirring at room temperatures the color turn to deep yellow immediately after addition of 20 L of NaOH then the solution is stirred for additional 10 min samples collected by centrifugation as mentioned above.

Example 10

(112) The treelike silver structures with multi branched edges like arms were synthesized according to above condition but using 1 mL of AgNO.sub.3, 2 mL of dextrose, added to 10 mL DDI water, then added 100 L of NaOH stirring with heating up to 100 C. the color turned to yellow to deep gray with precipitates after 3 min then the solution was allowed to cool to room temperature with stirring and samples collected by centrifugation as mentioned above.

Example 11

(113) The flower-like silver structures with more wide internal hollow like core, multi-edge like arms were synthesized according to above condition but using 2 mL of AgNO.sub.3, 1.0 mL of TSC, 1.0 mL of dextrose, then added 50 L of NaOH and stirring with heating up to 100 C. the color turned to yellow to deep yellow after 5 min then the solution was stirred till cooling to room temperature and samples collected by centrifugation as mentioned above.

Example 12

(114) The dendrimer silver structures were synthesized according to above conditions but using 0.5 mL of AgNO.sub.3, 0.5 mL of dextrose, added to 5.0 mL of DDI water, then added 100 L of NaOH with stirring at room temperature the color turned to yellow to deep gray and a precipitate was formed immediately, then the solution was stirred for additional 5 min and samples collected by centrifugation as mentioned above.

Example 13

(115) The unique flower silver structures with more internal hollow, rough surface were synthesized according to above conditions but using 1.0 mL of AgNO.sub.3, 1.0 mL of TSC, 1.0 mL of dextrose, then added 50 L of NaOH, then stirring with heating up to 100 C., the color turned to yellow to deep gray then the solution was stirred till cooling to room temperature, and samples collected by centrifugation as mentioned above.

Example 14

(116) The cube silver structures were synthesized according to above condition but using 0.4 mL of AgNO.sub.3, 0.4 mL of dextrose, 0.4 mL of TSC, added to 5 mL of DDI water, then added 200 L of ascorbic acid, then added 200 L of NaOH and stirring at room temperature the color turned to yellow to deep gray immediately, then the solution was stirred for additional 5 min and samples collected by centrifugation as mentioned above.

Example 15

(117) The pyramidal silver structures were synthesized according to above condition but using 0.5 mL of AgNO.sub.3, 0.5 mL of dextrose, added to 5.0 mL of DDI water, then added 300 L of NaOH stirring at room temperature the color turned to yellow to deep gray and a precipitate formed immediately, then the solution was stirred for additional 5 min and samples collected by centrifugation as mentioned above.

Example 16

(118) The bibode silver structures were synthesized according to above conditions but using 1.0 mL of AgNO.sub.3, 1.0 mL of dextrose, then added 150 L of NaOH and stirring at room temperature the color turned to deep gray immediately, then the solution was stirred for additional 5 min and samples collected by centrifugation as mentioned above.

Example 17

(119) The triangular silver structures were synthesized according to above conditions but using 1.0 mL of AgNO.sub.3, 1.0 mL of TSC, 1.0 mL of dextrose added 100 L of NaOH stirring with heating up to 100 C. the color turned to yellow to deep gray immediately after 5 min then the solution was stirred till cooling to room temperature and samples collected by centrifugation as mentioned above.

Example 18

(120) The octahedral multilayer silver structures were synthesized according to above condition but using 0.5 mL of AgNO.sub.3, 0.75 mL of dextrose, then adding 300 L of ascorbic acid, stirring at room temperature the color turn to deep gray immediately, then the solution was stirred for additional 5 min and samples collected by centrifugation as mentioned above.

Example 19

(121) The octahedral multi layer, with pores on the surface silver structures were synthesized according to above condition but using 0.5 mL of AgNO.sub.3, 0.75 mL of dextrose, then added 200 of ascorbic acid, stirring at room temperatures the color turned to deep gray immediately, then the solution was stirred for an additional 5 min and samples collected by centrifugation as mentioned above.

Example 20

(122) The ribbed silver structures were synthesized according to above conditions but using 0.5 mL of AgNO.sub.3, 0.75 mL of dextrose, then added 150 L of ascorbic acid, stirring at room temperatures the color turn to deep gray immediately, then the solution was stirred for additional 5 min and samples collected by centrifugation as mentioned above.

Example 21

(123) The synthesis of multi ribbed with cubes decorated on the surface silver structures was done according to above conditions but using 0.5 mL of AgNO.sub.3, 0.75 mL of dextrose, then adding 100 L of ascorbic acid, stirring at room temperatures the color turned to deep gray immediately, then the solution was stirred for an additional 5 min and stirrer turn off and samples collected by centrifugation as mentioned above.

Example 22

(124) The synthesis of octahedral with cube decorated on the surface silver structures was carried out according to above conditions but using 0.5 mL of AgNO.sub.3, 0.75 mL of dextrose, added to 10 mL DDI water, then adding 100 L of ascorbic acid and 100 L of NaOH directly after ascorbic acid and stirring at room temperature. The color turned to pale gray immediately, then the solution was stirred for an additional 5 min and samples collected by centrifugation as mentioned above.

(125) Synthesis of Silver Stars, Flower, Myriad Dendrimer and Butterfly Structures.

(126) Stars, flower, myriad dendrimer, butterfly silver structures fly have been synthesized by reduction of AgNO.sub.3 in aqua's TSC solution, TSC with polymer solution by ascorbic acid at room temperature. Likewise reduction of AgNO.sub.3 in aqueous TSC with polymers was achieved by UV and then by ascorbic acid. The particle morphology and size were manipulated by changing amounts of AgNO.sub.3, TSC, and duration of UV irradiation.

Example 23

(127) The silver stars with soft arms were synthesized by the chemical reduction of silver nitrate in aqueous phase. Briefly; 0.3 mL of 0.001-0.1 mM AgNO.sub.3, 0.5 mL of a 0.0001-2.0M TSC solution were added to 10 mL DDI, followed by 100 L of ascorbic acid (0.3-3.0 M) and stirred at room temperature. The color changed immediately to gray and the solution was stirred for an additional 5 min, and stirrer turn off. The varying amounts of TSC (0, 300, 400, 500, 600, 700, 800, 1100 L) lead to formation of silver stars with soft arms.

Example 24

(128) Silver stars with soft and rough, branched, more layer of arms and holes as core like were formed by the same conditions above but using (50, 100, 200, 300, 400, 500, 600, 700, 800 L) of 0.001-0.1 mM AgNO.sub.3 and using 0.5 mL of a 0.0001-2.0M TSC solution added to 10 mL DDI, then adding 100 L of ascorbic acid 0.3-3.0M, and stirred at room temperature. The color change immediately to gray. After color change, the solution was stirred for an additional 5 min. The color changed faster by using higher amounts of AgNO.sub.3.

Example 25

(129) Silver flower structures were formed by the same condition above but using 50, 100, 150, 200, 250, or 300 L of 0.3-3.0 M ascorbic acid added to 10 mL DDI containing 300 L 0.001-0.1 mM AgNO.sub.3 and using 0.5 mL of 0.0001-2.0M TSC solution, then stirred at room temperature. The color changed immediately to gray, the solution is stirred for an additional 5 min. The color changed faster by using higher amounts of ascorbic acid.

Example 26

(130) Silver myriad dendrimer structures were synthesized by the same conditions above and varying the amounts of TSC and AgNO.sub.3. Briefly 300, 200, or 100 L of 0.0001-2.0 M TSC were added to 300, 200, or 100 L of 0.001-0.1 mM AgNO.sub.3; and 10 mL DDI then 100 L of 0.3-3.0M ascorbic acid were added. After stirring at room temperature the color changed to gray then the solution was stirred for an additional 5 min. The color changed faster when higher amounts of TSC and AgNO.sub.3 were used.

Example 27

(131) Silver butterfly were synthesized by the same condition above but using 500 L of 0.0001-2.0 M TSC and 300, 200, or 100 L of 0.001-0.1 mM AgNO.sub.3 with 10 mL DDI then adding 80, 100 and/or 150 L of 0.3-3.0 M ascorbic acid. After stirring at room temperature the color changed to gray then the solution was stirred for an additional 5 min. The color changed faster when higher amounts of ascorbic acid and AgNO.sub.3.

Example 28

(132) Silver stars were synthesized by using 0.001-0.1 mM AgNO.sub.3, 0.5 mL of a 0.0001-2.0M TSC, 0.3 mL of polymer solution (PEG, PVA, PEI,) added to 10 mL DDI, then subjected to 18 W ultraviolet light at wave length (430 nm) for 30 min. The color turned to gray gradually. After adding 100 L of 0.3-3.0 M ascorbic acid at room temperature the gray color changed to deep gray immediately. The solution was stirred for an additional 5 min. PVA or PMMA have been used with TSC as morphology controlling gannet for synthesis of star like silver structures with branched arms. Briefly, 0.3 ml of polymer solution (PVA or PMMA) with mixed with AgNO.sub.3, TSC and reduced with ascorbic acid with and without UV (Claim 15). The varied time of UV irradiation have noted effects on the size and morphology of the synthesized particles.

Example 29

(133) Silver stars, were synthesized without UV irradiation by using 0.3 mL of a 0.001-0.1 mM AgNO.sub.3, 0.5 mL of a 0.0001-2.0 M TSC, 0.3 mL of polymer solution such as (PEG, PVA, PEI) added to 10 mL DDI. 100 L of 0.3-3.0M ascorbic acid was added and stirred at room temperature the gray color change gradually to deep gray immediately, after the color changed, the solution is stirred for an additional 5 min. PVA or PMMA have been used with TSC as morphology controlling gannet for synthesis of star like silver structures with branched arms. Briefly, 0.3 ml of polymer solution (PVA or PMMA) with mixed with AgNO.sub.3, TSC and reduced with ascorbic acid with and without UV (Claim 15). The varied amount of TSC, AgNO.sub.3, polymers have noted effects on the size and morphology of the synthesized particles.

(134) Zeta potential for silver structures (Example 1-7 and 23-29) were measured with dynamic light scattering NANO ZS Malvern zeta sizer equipment (Worcestershire, UK), at 25 C., using a HeNe laser of 633 nm wavelength and a detector angle of 173. Four independent measurements were made for each sample as tabulated in table 1. The result clearly demonstrated that, the silver structures are coated with negative charge ranged between 33 and 65 which it is strong enough to protect it from aggregation. Therefore the presence of electrical charge on the surface of silver structures makes it a promising tool in clinical diagnostics.

Example 30

(135) See Controlled synthesis and characterization of hollow flower-like silver nanostructures; attached to U.S. 61/594,817 and published as Eid & Azzazy, International Journal of Nanomedicine 2012:7 1543-1550 (Mar. 19, 2012).

(136) Only the polyol process was versatile for the synthesis of the following structures: spherical, cubes, pyramidal, hollow cubes, bars, rice-like, octahedral, beam and spheroid. Polyol method gives mixtures of structures and there is a need for separation of different structures by nanofiltration.

(137) Methods using poly (lactic-co-glycolic) acid templates gave similar star-like structures to the method of the invention but were not observed to provide the same variety of different structures. The method also requires a polymer template with or without UV irradiation and generates large size of stars, with lack of size control.

(138) The nanoparticles of the invention can be used in numerous different applications.

(139) They may be further processed, for example, by application of a surface coating to modify their solubility in polar or nonpolar media. For many biological applications, the application of a polar surface coating would be advantageous to provide solubility in aqueous media or in biological fluids. Nanoparticles may be functionalized with particular molecules to target them to particular receptors or to track their distribution. Examples of such targeting moieties include antibodies, protein or nucleic acid ligands that bind to specific receptors or molecules, aptamers, or other tags such as radioactive agents, fluorescent dyes, or tags like (strept)avidin or biotin or nickel and histidine. Hollow nanoparticles or nanoparticles having hollow portions can be used as vessels for carrying or containing other molecules such as those useful for imaging, plasmonics, or biosensing.

(140) Silver spherical nanoparticles have been encapsulated inside liposome nanoparticles to act as a sustained broad spectrum antibacterial agent. The kinetic release, cytotoxicity, and antibacterial against Gram negative and Gram positive Bactria namely; Escherichia coli, Salmonella enterica, Pseudomonas aeruginosa, and Staphylococcus aureus were investigated against as described in the paper [Sustained Broad Spectrum Antibacterial Effects of Nanoliposomes Loaded with Silver Nanoparticles]. Thee strains growth was inhibited by more than 80% upon usage of using 200-225 M of silver nanoparticles. The results obviously revealed that, the silver nanoparticles are promising tools in antibacterial therapy and wound healing. The optical properties of the silver nanoparticles were investigated by UVvisibleNIR measurements (Perkin Elmer UV/Vis/NIR Win lab lambda 950, 950N6102502, UK) and Pro Raman-L Analyser (Enwave Optronics Inc. 18200 McDurmott Calif. 92614, USA). The spectrum of the silver structures exhibit all characteristic peaks corresponding to different modes of plasmon excitation of all structures. There is more than dominant one band, located at around 342-300 nm and with intensity ranged between 500-13,000 a.u. Therefore the ability of silver structures to located band in both UV and NIR makes it a promising tool in SERS, optical, electronic, sensors and plasmonic applications.

Example 31

(141) Dextrose was used to reduce silver nitrate with heating to form spherical silver nanostructures. Briefly, the spherical morphology was synthesized by the chemical reduction of silver nitrate in aqueous phase. An amount of 0.001-2M AgNO.sub.3 solution mixed with 0.1-2 mM of dextrose and stirred with heating the color change immediately to deep yellow. After the color changed, the solution is stirred for an additional 5 minutes, and stirrer turn off. The particles collected by centrifuge at 1400 rpm for 10 minute, the supernatant can be removed, precipitate are re-suspended in DDI water and repeat three time for remove excess of dextrose. The Silver particle having size in the range of 10-50 nm and particle size depended on the concentration of dextrose.

(142) TABLE-US-00001 TABLE 1 Zeta potential for all silver structures samples 1-29. Examples Zeta potential mV 1 45 2 2 35 3 3 28 3 4 55 4 5 37 2 6 44 5 7 33 4 23 65 3 24 55 2 25 48 4 26 62 5 27 59 4 28 61 3 29 48 3

(143) TABLE-US-00002 TABLE 2 Other Versatile Methods for Synthesis of Silver Nanoparticles: Morphology Methods Reagents of Structures Reference Citrate Materials: Spherical, [1-7] reduction AgNO.sub.3 quasi sphere 2-propanol and, N.sub.2O octahedral., Sodium citrate (stabilizer) wire and pH controller such as NaOH triangle UV Polyol Materials Spherical, [8-25] (ethylene AgNO.sub.3 or (CF.sub.3COOAg) wires, beam, [26] glycol + PVP) Ethylene glycol (solvent- rice, cubes, reducing agent) bars, PVP (stabilizer polymer). bipyramid, Reducing agent: NaBr beam, HNO.sub.3 octahedron, NaHs with HCl cube, CuCl, FeCl.sub.3 truncated formamide/ethanol octahedron, and tubes Light- Material: Silver [7, 27-33] Mediated AgNO.sub.3 chains, Synthesis Methoxy polyethylene glycol polygonal or photosensitive polymer as plates, Template Ethanol solution disk, wire, Hyperbranched polyurethane rods and UV octahedron, Polyol reagent spherical Seed growth Materials: rods, wire, [6, 30, AgNO.sub.3 branched, 34-40] Sodium citrate, decahedron L-arginine, and cubes PVP NaBH.sub.4 Ascorbic acid PtCl or other metal salt CTAB or BDAC or combination of two

Example 32

(144) AgNPs Encapsulated in Nanoliposomes as Effective Broad Spectrum Anti-Microbial Agents.

(145) Nanoliposomes (<50 nm) were prepared using a modified reverse phase evaporation method and spherical dextrose-capped AgNPs were synthesized. The prepared liposome AgNPs (LAgNPs) were characterized and tested for their antibacterial effects. The size of LAgNPs is 25-80 nm. Release of AgNPs from nanoliposomes was sustained over 10 h. Complete growth inhibition of Escherichia coli, Salmonella enterica, Pseudomonas aeruginosa, and Staphylococcus aureus was achieved using 180, 200, 160, and 120 M, respectively, of LAgNPs. As shown below, LAgNPs exhibited sustained broad-spectrum antibacterial effects as compared to free AgNPs.

(146) Synthesis of spherical silver nanoparticles. Silver nitrate (AgNO.sub.3), dextrose, egg Phosphatidylcholine (PC), cholesterol (Cho), MacConckey agar medium, broth medium, agarized Czapek Dox, ethanol, chloroform, and sodium hydroxide (NaOH) were purchased from Sigma-Aldrich Chemie GmbH (Munich, Germany). HCl and HNO.sub.3 were purchased from El-Gomhouria Co, (Cairo, Egypt). Double deionized water (DDI) was prepared using a Milli-Q system (Direct-Q 3, Model ZRQSOPOWW, Millipore Corporation, Billerica, Mass.) with a resistivity of 18 Mcm.

(147) AgNPs were synthesized by the chemical reduction of AgNO.sub.3 in aqueous solution. Briefly, a round-bottom flask was cleaned thoroughly with aqua regia (3HCl: 1HNO.sub.3) then rinsed with DDI water. AgNO.sub.3 (0.01-1.0 M) was added to 0.1-2.0 M dextrose solution and dissolved in water at room temperature. NaOH (0.001-0.2 mM) was added during stirring. The solution changed from colorless to yellow, brown or green depending on the amount of AgNO.sub.3, dextrose, and NaOH. Following color change, the solution was stirred for an additional 5 min, centrifuged, and washed three times with DDI water to remove excess dextrose.

(148) Nanoliposomes were prepared using PC and Cho with different molar ratios (1:1, 1:2, 1:3, 1:4, 1:5, and 1:6). The lipid components were dissolved in 5 mL of chloroform and ethanol mixture (6:1, v/v). The solvents were removed using a rotary evaporator (Buchi RE-111 Rotavapor, Brinkmann, Westbury, N.Y.) at 55 C., 25 rpm, and high vacuum which has resulted in a lipid thin film. The film was redissolved in 10 mL PBS, pH 7.4 and the mixture was vortexed for 2 min and then sonicated using a probe sonicator (Model GM 2200, Bandelin Electronic, Berlin, Germany) with heating to form vesicles. The undispersed vesicles (aggregates) were separated by filtration.

(149) For the synthesis of LAgNPs, the thin films prepared above were rehydrated with 10 mL PBS, pH 7.4, containing AgNPs (5 nm). The mixture was vortexed and sonicated with heating and then incubated at room temperature for 3 h then filtered to remove aggregates and free unencapsulated AgNPs

(150) Mean particle size diameter and polydispersity index of nanoliposomes and LAgNPs were measured directly after synthesis, using photo correlation spectroscopy (Malvern Instruments Ltd, Worcestershire, UK).

(151) The size and morphology of the synthesized nanostructures were studied using scanning electron microscope (SEM, LEO SUPRA 55; Carl Zeiss AG, Oberkochen, Germany) and transmission electron microscope (TEM, JEOL X100, Japan). Briefly, silver samples were mounted on silicon wafer coated with aluminum foil and left 2 hours to dry before imaging without sputter coating before SEM imaging at an accelerating voltage of 6 kV and magnification of 150-200 k X. Liposome samples were diluted with BPS and sonicated for 3 min then negatively stained with 2% uranyl acetate and mounted on TEM grids (carbon film supported by a copper grid) and allowed to dry for 2 h before imaging with TEM at an accelerating voltage ranging from 200-220 kV and magnification of 400-500 kX. The particle size was reported as the mean diameter of randomly selected structures.

(152) The encapsulation efficiency of AgNPs in liposome was measured using atomic absorption spectrophotometer (Z-5000, Hitachi, Ltd., Tokyo, Japan). Briefly, 5 mL of the synthesized LAgNPs were injected into the system and the percentage of encapsulated AgNPs was calculated as follows: Silver loading=[(WTWS)/WT]100%, where WT is the total AgNPs added to the liposomes and WS is the portion of AgNPs that was not encapsulated and present in the supernatant after ultracentrifugation of LAgNPs.

(153) Different preparations of LAgNPs (PC: Cho; 1:1, 1:2, 1:3) loaded with AgNPs (5 nm) were placed in cellulose dialysis bags. The bags were suspended in 30 mL of PBS, pH 7.4 where AgNPs were released into the buffer by diffusion. The release of AgNPs from the dialysis bags was observed over a period of 10 h. Each hour, 1 mL of the buffer was removed, and substituted with fresh buffer, to measure the concentration of released AgNPs using atomic absorption spectrophotometer equipped with silver lamp (Z-5000, Hitachi, Ltd., Tokyo, Japan). The instrument parameters were: 328.1 nm wavelength; 5 mA lamp current; 0.5 nm band pass, fuel flow rate 0.9-1.2 L/min, and temperature of 1100 C.

(154) Escherichia coli, Salmonella, P. aeruginosa, and S. aureus were obtained from the department of Microbiology, VACSERA, Cairo, Egypt. Bacterial cells were cultured for 24 h on a MacConckey agar plates at 37 C. Colonies were resuspended in LB broth medium to achieve 10.sup.6 CFU/mL. This has been confirmed by measuring bacterial growth as optical density using a microplate plate reader (Tecn Infinity M200, CA, USA). Different concentrations of free AgNPs, LNPs (1PC:3Cho), or LAgNPs (1PC:3Cho:1AgNP) were added to the bacterial cultures. The bacterial growth in the culture medium was monitored by measuring the optical density at 700 nm.

(155) The optical density of bacterial cultures, grown in 3 replicates with shaking at 37 C. with and without nanoparticles, was recorded every 60 min. The rate of bacterial growth was calculated using the following formula: [(NtNo)/No100]; where N.sub.o was the OD of bacteria at time zero and N.sub.t is the OD of bacteria at the indicated time point.

(156) Table A illustrates particle size and polydispersity of nanoliposomes and LAgNPs using photon correlation spectroscopy.

(157) TABLE-US-00003 LNPs Size Poly LAgNPs Size Poly PC:Cho (nm) Dispersity (nm) Dispersity 1:1 240 35 0.13 25 4 0.01 1:2 270 25 0.16 45 5 0.015 1:3 310 22 0.18 50 3 0.02 1:4 340 38 0.2 65 1 0.04 1:5 410 40 0.31 70 3 0.05 1:6 450 28 0.4 80 5 0.07 *Same molar ratio of AgNPs was added to all PC/Cho preparations.

(158) The LAgNPs size ranged between 25 to 80 nm and the size of nanoliposomes between 250 to 400 nm. The SD values for LAgNPs were calculated from four independent measurements. AgNPs of 51 nm were used. Nanoparticles showed low polydispersity indices (Table A). The dynamic light scattering showed a narrow size distribution of LAgNPs and poly size distribution for nanoliposomes. The encapsulation of AgNPs into nanoliposomes has led to long-term stability of LAgNPs.

(159) The zeta-potential measurements of LAgNPs in solution ranged between 76 and 58 mV. After 3 months of storage at room temperature, measurements ranged between 69 and 45 mV. The persistence of the negative charge on the LAgNPs is indicative of their stability. The individual measurement results are shown in the supplementary data file.

(160) The size and shape of the synthesized AgNPs were studied using SEM and particle size was reported as mean diameter of randomly selected structures. Spherical silver nanostructures with size in the range of 51 nm were observed. UV spectroscopy of AgNPs with maximum absorbance at 410 nm was performed. SEM (A-C) of nanoliposomes that were prepared by using 1:1, 1:2, and 1:3 molar ratio of PC:Cho were obtained. The particles have spherical morphology with particle size in the range of 250-400 nm.

(161) The LAgNPs were analyzed by TEM and had spheroid shape with an average particle size between 25-90 nm. The addition of AgNPs to nanoliposomes may have contributed to the reduced size of nanoliposomes to less than 50 nm. The presence of charged AgNPs on the surface of nanoliposomes, supported by zeta potential measurements of AgNPs (38 mV) and LAgNPs (76 mV) (supplementary data), contributed to the stability of LAgNPs. This result is in agreement with previous reports which demonstrated that the presence of charged particles on the surface of nanoliposomes contribute to their stability [21-22]. Other groups used polymers or surfactants for the stabilization of liposomes [21-23].

(162) The amounts of AgNPs encapsulated into six different formulas of nanoliposomes 1:1, 1:2, 1:3, 1:4, 1:5, and 1:6 (PC:Cho) were 60%, 75%, 88%, 80%, 78%, and 70%, respectively. The highest encapsulation (88%) was achieved using nanoliposomes of 1PC:3Cho.

(163) The release of kinetic of free AgNPs from LAgNP prepared using 1:1, 1:2, and 1:3 PC:Cho was observed. The rate of release of AgNPs obeyed zero order kinetics with r.sup.2>0.96. This is similar to the release patterns of other drugs from liposomes. About 80% of the free AgNPs diffused out of the cellulose bag after 10 h. Release of AgNPs was 76%, 64%, 58% from liposomes prepared using 1:1, 1:2, 1:3 PC:Cho, respectively. Therefore, sustained release of AgNPs was observed using LAgNPs (1PC:3Cho; 50 nm). Because of their high encapsulation efficiency and sustained release of AgNPs, LAgNPs prepared using 1PC:3Cho were tested for their anti-bacterial effects.

(164) The antibacterial activity of LAgNPs prepared using 1PC:3Cho was assayed against four common bacterial species namely: E. coli, Salmonella, P. aeruginosa, and S. aureus and monitored by optical density measurements using a micro-plate reader. Different concentrations of the LAgNPs (20-225 M) were tested. LAgNPs completely inhibited bacterial growth of E. coli (180 M), Salmonella (200 M), P. aeruginosa (160 M), and S. aureus (120 M). The growth of the above strains was not completely inhibited using 200-225 M of AgNPs. Adding free nanoliposomes up to 150 M had no effect on bacterial growth then the growth decreased by 30-45% upon increasing the concentration of nanoliposomes to 225 M.

(165) LAgNPs were more effective in inhibiting growth of Gram positive bacteria (S. aureus) as compared to Gram negative bacteria (E. coli, S. enterica, and P. aeruginosa). This may be due to the difference in the structure of the outer walls of Gram positive and negative bacteria.

(166) Cytotoxicity of LAgNPs (0.05-0.3 mg/mL) to cultured human fibroblast cells was investigated using MTT assay. LAgNPs concentrations between 0.05 to 0.10 mg/mL were safe and did not affect cell viability; higher LAgNPs concentrations (0.2-0.3 mg/mL) were toxic to cultured fibroblasts.

(167) This example shows for the first time that nanoliposomes loaded with AgNPs are useful as broad-spectrum anti-bacterial agents. The modified reverse phase evaporation method allowed the preparation of nanoliposomes with size between 25-80 nm without the need for high pressure homogenizer and extruder. Also, the AgNPs were prepared by a green method and all reagents used for preparation of nanostructures were non-toxic. The presence of AgNPs stabilized the nanoliposomes and led to narrow size distribution of LAgNPs. LAgNPs prepared with 1PC:3Cho demonstrated high encapsulation capacity and provided long term release of AgNPs. The results show the feasibility of using LAgNPs as a new generation of antibacterial agents for sustained killing of multiple kinds of bacteria. The LAgNPs are stable in pharmaceutical preparations and provide long term anti-bacterial effect at the infection site.