Chemistry

Ultrasensitive detection of protein targets, using nanoparticle probe technology, entails:

• The introduction of serum from a clinical sample into a single-use, proprietary Test Cartridge;
• The protein of interest in the serum is then captured via simultaneous hybridization to:
  • Magnetic particles loaded with primary antibodies specific to the protein of interest, and
  • Gold nanoparticle probes co-loaded with secondary antibodies specific for the protein of interest and with hundreds of identical carrier oligonucleotides.
  • The result is a complex where the protein of interest is bound between a magnetic particle and a gold nanoparticle probe functionalized with hundreds of identical oligonucleotides.  
• The sample is then enriched, for the protein of interest, by applying a magnetic field to the sample (capturing the complex defined above), drawing off the residual supernatant, and washing away uncaptured materials; 
• The complexes are then released from the magnetic field and resuspended in a buffer that facilitates release (via increased temperature) of the identical barcode oligonucleotides from their gold nanoparticles;  
• The free identical oligonucleotides are then resuspended in a solution and captured and detected in the same sequence-specific DNA detection format (an array) as described above for DNA targets.  
• The quantitative analysis of results (array reading) is then performed on the Verigene^® Reader, with comparison of the result intensity to a standard curve for the protein of interest.

Multiplexing of multiple protein targets in a single clinical sample is possible by changing the primary antibodies on the magnetic particle and the secondary antibodies on the gold nanoparticle and functionalizing the gold nanoparticles with uniquely-sequenced oligonucleotides that have different base sequences to identify each protein target in the sample. A sample with multiple protein targets of interest, therefore, can have all of the proteins' levels measured by loading the sample with multiple magnetic bead-types, each with distinct primary antibodies for one of the protein targets, and by loading the sample with multiple gold nanoparticle-types, each with distinct secondary antibodies for one of the protein targets and a unique set of oligonucleotides for one of the protein targets. The result is multiple complexes, where each protein of interest is bound between its unique-magnetic particle and its unique gold nanoparticle probe that is functionalized with hundreds of unique oligonucleotides for its specific protein. The process defined above for a single protein is then followed. Once the oligonucleotides are released from each type of gold nanoparticle, the multiplexed slide array, containing multiple spots of different complementary capture oligonucleotides that bind with the specific complementary target oligonucleotide for each protein target, is exposed to the olignucleotide-rich solution for hybridization with specific spots for each protein of interest. The slide is then processed and read by the Verigene^® Reader.

• The sensitivity of this technology for low-abundance target protein detection is 2-3 orders of magnitude higher than that of ELISA-based technologies because the method incorporates two means for signal amplification:
  • The amplification of each protein molecule of interest into hundreds of corresponding identical oligonucleotides specific for a given protein; and
  • The amplification of a single oligonucleotide into a signal-enhanced gold nanoparticle (as described in the Direct Detection of Nucleic Acids section).