analyte dx

The 2PG MoM™ can detect different target analytes across the molecular spectrum. Nanopore sensors read individual molecules, one at a time, using electrical sensing and 3D synthetic DNA scaffolding reagents.

The 2PG MoM™ is an open system platform that allows independent developers to create assays for molecular and other diagnostic tests. See the Partners page for more information.

Voltage pulls molecules through, impeding current by a measured amount.

Nanopore sensors read individual molecules, one at a time, using electrical sensing. No optics. No chemistry.

Single molecule “event” has depth and duration, proportional to its size.

Matrix contains DNA, RNA, proteins, small molecules, resulting in many different event signatures.

Detecting Target Analytes

2PG's reagent compound

  • Bulk-phase association kinetics in 3D volume is faster and more efficient than 2D (surface) binding in ELISA tests.
  • 2PG’s reagent is a synthetic DNA scaffold tethered to a target analyte binding domain, derived from a 3rd party reagent (e.g., existing ELISA kit).
  • When scaffolds pass through the nanopore, they create discrete impedance signatures, depending on whether the target analyte is bound.

Types of Binding Molecules

Peptides:

Pros:

  • Easily conjugates to other molecules (e.g. DNA binding domains)
  • small, inexpensive and fast to (mass) produce.
Note: KD must be sufficient to achieve at least a minimum number of bound scaffolds—this establishes the lower limit of detection. Beyond this minimum, 2PG’s mathematical framework can quantitate original target concentration from sample, regardless of KD.

Aptamers:

Pros:

  • Can be synthetically produced to contain conjugation sites
  • Generally small

Cons:

  • Aptamer availability may be limited
  • May need serum stabilization (for blood)

Fab fragments:

Pros:

  • Good choice when engineered to contain a terminal group allowing conjugation (e.g. reduced cysteine)
  • Generally small
  • High affinity
Cons:
  • Must know original antibody sequence
  • Requires molecular engineering and protein expression expertise to make in sufficient quantities

Full length antibodies:

Pros:

  • Easily ported from existing assays “as-is”
Cons:
  • Since conjugation is generally done through free lysines, a fair amount of antibody needs to be available for development work
  • Large, so signal can dilute target. Sandwich or competition assay can be used to create more bulk and boost signal

Controlled Demonstration

Test: Target analyte (HIV antibody) spiked into saliva at clinical concentrations (nM)

Target is spiked into saliva and incubated with detection reagent, and read directly by nanopore sensor. No prep necessary.

Raw Data: Events are plotted based on depth and duration

Target bound complexes are detected by the pore for 10-30 seconds. Bound and unbound detection reagent events are used to calculate a quantitative result.

Target Quantitation: 3 parameter model is used to estimate target concentration

Metric Value
TTR Seconds to 5 mins.
LOD 1 pore (demonstrated) 500 pM (TTR minutes)
LOD 10 pore (projected) 1-10 pM

SANDWICH ASSAYS BOOST SIGNAL OF SMALL ANALYTES AND INCREASE SPECIFICITY

  • A secondary molecule such as an antibody improves the specificity of total assay. This is useful if the primary binder has off-target binding errors.
  • If the target is small, secondary molecule (e.g. antibody), increases bulk, making for better detection.

15 kDa protein with and without sandwich:

  • The bulked-small protein (green) was detected (99% conf) in 60 seconds of recording, compared to 10 minutes required without the bulk (red).
  • Enables quantitation at lower concentrations for improved LoD.

COMPETITION ASSAY FOR SMALL MOLECULES

Target small analyte competes with a payload-tethered competitive inhibitor

  • Events are plotted based on depth and duration
  • Maximum area (depth*duration) criteria used for positive detection of presence of small molecules