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Applications of Conjugated Polymer Nanoparticles in western blot and lateral flow point-of-care diagnostics

Rasikh Raza

Rasikh Raza, Sales lead

Conjugated Polymer Nanoparticle™ properties

Conjugated Polymer Nanoparticles (CPNs™) are fluorescent nanoparticles with a range of unique properties that can significantly benefit many life science applications (1). Each CPN™ contains a semiconductor light-emitting polymer core – containing iron oxide – encapsulated within a biocompatible surfactant, permitting a range of surface chemistries for targeting molecule conjugation. CPNs™ demonstrate intense fluorescence across emission wavelengths ranging from 420 to 1130 nm, with consistent brightness and size (approximately 80 nm), as can be seen under a super-resolution microscope (Figure 1).

Figure 1: CPN™ 420 (Violet), CPN™ 435 (Indigo) and CPN™ 550 (Yellow) imaged using super-resolution microscopy at the National Physical Laboratory, UK, displaying consistent size and brightness.

The properties of CPNs™ allow them to enhance a range of life science applications, where they provide benefits over conventional fluorophores.  CPNs™ are intensely bright, with experimental data finding them to be 100-1000 times brighter than Q-dots and Q-beads (Figure 2). Further experimental data has shown that they are stable across wide ranges of temperature and pH (2), emitting consistent fluorescence over time, showing no decrease in brightness after 24 months stored in ambient conditions (Figure 3).

In addition, due to their iron oxide core, CPNs™ are able to be manipulated with a magnet, offering potential for target separation within a sample, as well as increased speed in assays. A range of conjugation protocols and surface chemistries are available, such as carboxyl, maleimide-thiol, alkyne click chemistry and streptavidin for biotinylated linkage to a variety of targeting molecules, including antibodies, oligonucleotides and receptor ligand proteins. This allows CPNs™ to be used in diagnostic techniques (3) such as western blot and lateral flow, where their properties provide enhanced results over current methods.

Figure 2: Experimental data from the Ozcan Research Group, University of California, Los Angeles, showing that CPN™ 610 (Orange) is up to 100-1000x brighter than equivalent Q-beads and Q-dots. Data measured on a standard ELISA plate reader with excitation and emission optimised for each fluorophore.

Figure 3: CPNs™ remain photostable for up to 24 months, as shown here, CPNs™ retain their fluorescence after a long period of storage.

CPN™ benefits to diagnostic techniques

CPNs™ offer advantages in diagnostic techniques such as western blot, lateral flow and ELISA due to the unique properties outlined above.  As a result of their powerful brightness, even low levels of the target molecule produce a clearly visible signal, improving detection sensitivity across a wide range of assay formats and reader systems (Figure 4).

Further, CPNs™ deliver robust and stable fluorescence even at low concentrations (Figure 4) that won’t fade over time, allowing the results to be easily stored. In addition, the range of CPN™ emission wavelengths available supports the detection of several target molecules simultaneously, offering improved efficiency in testing.

Another key benefit is CPN™ stability in ambient conditions, allowing for both long periods of storage and use outside of the lab, crucial for point-of-care diagnostics. These properties not only improve functionality and ease-of-use, but they can also offer improved detection over diagnostic markers currently available.

Figure 4: detection sensitivity offered by CPNs™. A. Reader detection limit based on calibrated intensity of CPN™ signals at a range of CPN™ quantities. B. CPN™ fluorescence at a range of concentrations. C. CPN™ fluorescence at a range of concentrations, detected using a SpectraMax 2e reader system (Molecular Devices). D. CPN™ signal brightness from a range of quantities of CPN™. E. Test line brightness at a range of CPN™ concentrations.

CPNs in western blot

CPNs™ can be used in western blot analyses to detect a protein of interest, as shown in Figure 5. In this example, the protein sample was prepared and transferred to the membrane with the primary antibody-linked CPNs™, which were then incubated at room temperature for 30 minutes. CPNs™ are sufficiently robust to label size markers on gel and be transferred to the membrane. The western blot signal, visualised by a gel imaging system, shows that CPNs™ can produce a strong and robust signal, allowing clear identification of the protein or proteins of interest, visible even after storage for extended periods of time.

Figure 5: A. CPN™ western blot result using anti-mouse conjugated CPN™ 510B (green) to detect a mouse-anti-CD44 protein sample.  B. CPN™ western blot result using anti-mouse conjugated CPN™ 900 (IR-I) to detect a mouse-anti-CD44 protein sample. Signal visualised by ChemiDoc (BioRad).

Figure 6: A. Multiplexed lateral flow detection strips using CPNs™ to detect two antibodies simultaneously. B. Comparison of test line brightness between CPN™ 610 (Orange) and conventional fluorophore Dylight™ under matching conditions.

CPNs™ in lateral flow rapid diagnostics

The benefits of CPNs™ outlined above are also evident in lateral flow assays (4, 5). This includes their intense brightness when compared to alternative fluorophores (Figure 6 B), and the use of differently coloured CPNs™ in multiplexed lateral flow assays (Figure 6 A). As a result, CPNs™ offer several significant benefits over conventional fluorophores in lateral flow assays. These benefits can be further enhanced with the use of a reader device, detecting a positive signal from as few as 600 CPNs (Figure 4 A).

Lateral flow assays are often used in point-of-care diagnostics due to their ease-of-use and the rapid results they can provide (4, 6). In addition, they can produce high levels of accuracy from a robust, cost-effective, test, making them well-suited to widespread use outside of the lab. Based on their ability to enhance lateral flow assays and significantly increase sensitivity CPNs™ have prominent applications in rapid diagnostic tests (RDTs).

The use of CPNs™ in RDTs may play a role in responding to many current and future healthcare challenges. By linking CPNs™ to the appropriate antigen, virus detection is enabled. CPNs™ give a strong visible signal without a reader, but the incorporation of a reader device would facilitate PCR levels of sensitivity and quantitative results (Figure 4 A). In conjunction with a portable reader, CPN™-based viral diagnostics will achieve levels of sensitivity previously only possible within a lab. As discussed above, thanks to the unique properties of CPNs™, this technology will allow detection of low viral levels whilst being sufficiently robust and stable enough to be used at a variety of locations.

Work is currently underway to use this technology for a COVID-19 diagnostic test, identifying both symptomatic and asymptomatic carriers within ten minutes. In addition, the testing platform under development has the potential to detect other diseases, along with applications in agriculture and food testing.

Summary

As a result of their properties, CPNs™ can deliver enhanced sensitivity, improved stability and rapid, reliable results across a range of life science assays, including lateral flow and western blot. This innovative technology will not only provide benefits in the lab, but will allow the development of portable, point-of-care rapid diagnostic tests to help meet current and future healthcare needs. CPNs™ have the power to enhance the field of rapid diagnostics, providing clear improvements over what is currently available.

References

  1. Tuncel, D. and Demir, H., 2010. Conjugated polymer nanoparticles. Nanoscale, 2(4), p.484.
  2. Chan, Y. and Wu, P., 2014. Semiconducting Polymer Nanoparticles as Fluorescent Probes for Biological Imaging and Sensing. Particle & Particle Systems Characterization, 32(1), pp.11-28.
  3. Xu, X., Liu, R. and Li, L., 2015. Nanoparticles made of π-conjugated compounds targeted for chemical and biological applications. Chemical Communications, 51(94), pp.16733-16749.
  4. Swanson, C. and D'Andrea, A., 2013. Lateral Flow Assay with Near-Infrared Dye for Multiplex Detection. Clinical Chemistry, 59(4), pp.641-648.
  5. Juntunen, E., Myyryläinen, T., Salminen, T., Soukka, T. and Pettersson, K., 2012. Performance of fluorescent europium(III) nanoparticles and colloidal gold reporters in lateral flow bioaffinity assay. Analytical Biochemistry, 428(1), pp.31-38.
  6. Chen, Y., Chen, Q., Han, M., Liu, J., Zhao, P., He, L., Zhang, Y., Niu, Y., Yang, W. and Zhang, L., 2016. Near-infrared fluorescence-based multiplex lateral flow immunoassay for the simultaneous detection of four antibiotic residue families in milk. Biosensors and Bioelectronics, 79, pp.430-434.

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