Direct Detection of Nucleic Acids

Chemistry

The video animation shown above is provided for illustration / education purposes only. It is not intended to depict the exact format or function of the proprietary gold nanoparticle technology that Nanosphere employs in any of its diagnostic or for research use only products.

The detection of DNA or RNA targets using nanoparticle probe technology entails: 

• Automated nucleic acid extraction and PCR amplification (for some assays) from a clinical sample on the Verigene® Processor SP
• Automated transfer of eluted nucleic acids or amplicons into a single-use, proprietary Test Cartridge. The DNA is then sheared, via a proprietary sonication process, into 300-500 base-pair fragments in order to facilitate hybridization in subsequent steps.
• In the initial hybridization step, the sheared genomic DNA (target nucleic acid) is simultaneously hybridized to:
  • Single-stranded DNA capture oligonucleotides arrayed in replicate on a solid support (an array), and
  • Sequence-specific mediator oligonucleotides that detect single-copy DNA regions of each target of interest.
  • A washing step then follows that removes unhybridized gold nanoparticle probes.
• Silver signal amplification is then performed on the gold nanoparticle probes that are hybridized to captured DNA targets of interest.
• There then follows a washing step to remove unreacted signal amplification reagents.
• Qualitative analysis of results (slide reading) can now be performed on the Verigene^® Reader.

General Description

• Single use, self-contained test unit comprised of the following:
  • Reagent Pack — single use plastic carrier that creates a hybridization chamber on the surface of the array, houses all of the reagents needed for a single Test, and captures all of the waste materials generated during Test processing.
  • Substrate Holder (an array) — glass slide, solid support for the capture oligonucleotides required by the Test.
• Each Test Cartridge is designed for single-use analysis of one sample.

Functionality

• The Test Cartridge houses all of the reagents necessary for Nanosphere's proprietary nanoparticle-detection chemistry, which consists of:
  • Fragmentation and denaturation of genomic DNA or RNA via on-board sonication.
  • Primary hybridization of genomic DNA or RNA to capture probe oligonucleotides and mediator oligonucleotides under conditions of sufficient stringency. 
  • Washing away of unbound sample nucleic acids and mediator oligonucleotides.
  • Secondary hybridization of gold nanoparticle probes to captured target nucleic acid / mediator oligonucleotide complexes.
  • Washing away of unbound gold nanoparticle probes with oligonucleotides.
  • Signal amplification of gold nanoparticles oligonucleotide probes.
  • Washing away of unreacted signal amplification reagents.

Applications

The direct detection of single or multiple nucleic acid targets with single-base pair resolution has diagnostic applications in several clinical areas, including:

• Infectious Diseases
• Personalized Medicine
• Cardiology
• Human Genetics