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. 2022 Mar;113(3):e23481.
doi: 10.1002/bip.23481. Epub 2021 Nov 23.

Cyclopentane peptide nucleic acid: Gold nanoparticle conjugates for the detection of nucleic acids in a microfluidic format

Harsha Amarasekara  1 , Kaylyn M Oshaben  1   2 , Kendra B Jeans  1 , Paniz Rezvan Sangsari  3 , Nicole Y Morgan  3 , Brian O'Farrell  2 , Daniel H Appella  1
Affiliations

Cyclopentane peptide nucleic acid: Gold nanoparticle conjugates for the detection of nucleic acids in a microfluidic format

Harsha Amarasekara et al. Biopolymers. 2022 Mar.

Abstract

Routine patient testing for viral infections is critical to identify infected individuals for treatment and to prevent spreading of infections to others. Developing robust and reliable diagnostic tools to detect nucleic acids of viruses at the point-of-care could greatly assist the clinical management of viral infections. The remarkable stability and high binding affinity of peptide nucleic acids (PNAs) to target nucleic acids could make PNA-based biosensors an excellent starting point to develop new nucleic acid detection technologies. We report the application of cyclopentane-modified PNAs to capture target nucleic acids in a microfluidic channel, and the use of bioorthogonal PNAs conjugated to gold nanoparticles as probes to semi-quantitatively signal the presence of a target nucleic acid derived from HIV-1. The basic results presented could be used to develop more advanced devices to detect nucleic acids from viruses such as HIV, SARS-CoV-2, and a wide range of other human diseases.

Keywords: Peptide nucleic acid; gold nanoparticles; microfluidics; nucleic acid detection.

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Conflict of interest statement

Conflicts of interest

D.A., K.O., and B.O.F are co-inventors on a patent application covering portions of the work, as filed by the U.S. Government (US2021021147) and Altratech under a Collaborative Research and Development Agreement. B.O.F is a founder, director, and shareholder of Altratech.

Figures

Figure 1.
Figure 1.
Comparison of nucleic acids (DNA/RNA), standard aegPNA, and tcyp-modified PNA; *stereogenic centers that can be manipulated to specify the handedness of the helix; B = nucleobase (C, T, A, or G).
Figure 2.
Figure 2.
Specificity of PNA binding depends on the stereochemistry of tcyp modified PNA monomers.
Figure 3.
Figure 3.
UV-Testing data for AuNP-PNA-A (20 nm) Conjugate; λmax = ~ 520 nm. Experiments were conducted over 120 min; time = 0, 5, 10, 15, 30, 60, and 90 are presented in the graph.
Figure 4.
Figure 4.
Signal intensity generated in the microfluidic channel by the silver developer across the range of RNA concentrations examined. The intensity value at [RNA] = 0 is the background of the channel.
Scheme 1.
Scheme 1.
Protocol of Nucleic-Acid-Detection Microfluidic Assay. 1: Introduction of target nucleic acid sequence to the microfluidic device; 2: Attachment of AuNP-PNA conjugates to target nucleic acid via the RP-PNA; 3: Color development using silver color enhancer solutions A and B.
See this image and copyright information in PMC

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