Nanopixels: The Breakthrough Enabling Ultra-High Multiplex PCR

How Hyperplex PCR (hpPCR) overcomes the traditional limits of multiplex PCR to enable high-precision detection of dozens to hundreds of DNA targets in a single reaction.

How Nanopixels Enable Scalable Multiplexing in hpPCR

Multiplexing in molecular assays is usually limited by detection.

Most PCR systems distinguish targets using fluorescent colors. Each assay is assigned a specific fluorophore, and instruments typically detect only a small number of colors, often four to six. As a result, the number of targets that can be measured in a single reaction is inherently limited.

This is one of the fundamental reasons most PCR assays remain limited to small multiplex panels.

Nanopixels are optically encoded nanoparticle probes that form the detection backbone of Hyperplex PCR™ (hpPCR™). By enabling combinatorial optical encoding of targets, Nanopixels allow hpPCR to measure dozens to hundreds of biomarkers in a single reaction well without expanding the number of colors.

The limits of color-based multiplexing

Traditional PCR instruments typically provide only four to six fluorescence channels. This is because fluorophores do not emit at a single exact wavelength, but across broad emission spectra.

As more colors are added, the signals become harder to separate cleanly using filters and detectors, increasing cross-talk between channels and placing a practical limit on how many fluorophores can be used reliably in the same assay.

A different approach: optical encoding

hpPCR overcomes the detection limits of traditional PCR by using Nanopixels — optically encoded nanoparticle probes that enable scalable multiplex detection.

Instead of assigning one fluorescent color per assay, hpPCR generates microscopic amplification products that are labeled with encoded Nanopixels. Each Nanopixel carries a distinct optical signature that uniquely identifies a target molecule.

Because these signatures are encoded optically rather than tied to individual fluorescence channels, the number of distinguishable targets is no longer constrained by the number of colors available in the instrument.

This encoding strategy enables combinatorial scaling of detection, allowing dozens to hundreds of molecular targets to be distinguished within a single reaction well.

In practice, Nanopixels act as a universal detection layer for hpPCR assays. New targets can be added by assigning a new optical code rather than introducing additional fluorescent channels, enabling multiplex panels to grow without fundamentally changing the detection system.

Why nanoparticle probes matter

Nanoparticle probes offer several advantages for multiplex detection:

• Precise tuning, the physical size enables high degree of color control

• High optical brightness, enabling robust detection

• Fluorophore protection, improving assay reproducibility and data quality

• Flexible encoding schemes, supporting large multiplex panels

Because Nanopixels are universal probes rather than assay-specific fluorophores, they also simplify panel expansion. New targets can be added without redesigning the entire detection system.

Multiplexing without redesigning the instrument

By moving from fluorophore-based detection to optically encoded nanoparticle probes, multiplexing is no longer constrained by the number of fluorescence channels.

Instead, multiplexing becomes a function of encoding capacity.

This shift enables hpPCR to achieve multiplex levels that exceed conventional PCR approaches while maintaining reliable detection and quantification.

Expanding what PCR can measure

High multiplex capability changes what PCR workflows can accomplish.

Instead of measuring a few targets at a time, laboratories can analyze large panels of mutations, genes, or biomarkers within a single reaction.

Nanopixel optical encoding is one of the key technologies that enables this shift—transforming multiplex PCR from a limited capability into a scalable measurement platform.

Want to see in-depth Nanopixel data and performance? Download our technology note here.