Direct detection of disease biomarkers in clinical specimens using cationic nanoparticle-based assays & 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.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS[0001]This application claims priority under 35 U.S.C. §119(e) to U.S. Provisional Application 61 / 593,019, filed Jan. 31, 2012, and to U.S. Provisional Application 61 / 594,817, filed Feb. 3, 2012, each of which is hereby incorporated by reference in its entirety.BACKGROUND OF THE INVENTION[0002]1. Field of the Invention[0003]Methods for detecting pathogens using cationic gold- or silver-nanoparticles. The assays can detect amplified and / or unamplified RNA and / or DNA from viruses, bacteria and other organisms, such as Hepatitis C virus and mycobacterium tuberculosis. The invention is also directed to anisotropic silver nanoparticles and methods for synthesizing and using them.[0004]2. Description of the Related Art[0005]Cationic gold nanoparticle based assays. Many molecular diagnostic assays are commercially available for the detection of several viral / bacterial nucleic acids (DNA / RNA) in patients' blood. Although these assays have high sensitiv...

AI Technical Summary

Benefits of technology

The present invention solves problems associated with the use of gold nanoparticles for detection of target molecules. It detects unknown target molecules in a biological sample without the need for hybridization to a probe on a solid support. This avoids the problem of false positives caused by aggregation of cationic gold nanoparticles. The invention uses silica or magnetic nanoparticles that are attached to a specific probe or capture ligand by a crosslinker to isolate or purify the target molecule from a solution containing many molecules. This avoids the problem of false positive readings due to aggregation of cationic gold nanoparticles with non-target nucleic acids or contaminants. The invention provides a simple and homogeneous based assay that can be used outside of a hospital or medical laboratory to detect the target molecule visually by the naked eye without equipment like a scanner. The color change produced by the aggregation of cationic gold nanoparticles in the presence of a target molecule is directly proportional to the target concentration in a biological sample. Quantification does not require the use of equipment often unavailable in the field, such as a spectrophotometer. The invention is also directed to a method of detecting a material or molecule of interest by contacting a sample with positively charged gold and / or silver nanoparticles, determining the aggregation of nanoparticles after contacting them with the sample, and detecting the nucleic acid or protein in the sample when the nanoparticles aggregate in comparison with a control sample that does not contain the nucleic acid or protein to be detected.

Problems solved by technology

Although these assays have high sensitivity and specificity, most of them are time consuming, labor intensive, expensive, and require specialized equipment.

A significant drawback to the prior art methods which use gold nanoparticles is that the presence of a capture probe in the absence of a target molecule leads to aggregation of the gold nanoparticles because the capture probes bind to the cationic gold nanoparticles via their phosphate backbones.

Prior art methods require extra steps and labor are required to avoid this problem.

Another problem with prior methods is the requirement for equipment that is often not available under field conditions, such as the need to use a scanner to detect hybridization of a target molecule to a capture probe or ligand or the requirement for use of a laboratory spectrophotometer to quantify color change produced by aggregation of gold nanoparticles.

The difficulties encountered in fabricating anisotropic, controlled hollow nanostructures include high cost, the use of toxic chemicals, and expensive and complex laboratory equipment requiring highly skilled technicians.

Images