Conductive Ink-Coated Paper-Based Supersandwich DNA Biosensor for Ultrasensitive Detection of Neisseria gonorrhoeae

Niharika Gupta; D Kumar; Asmita Das; Seema Sood; Bansi D Malhotra

doi:10.3390/bios13040486

Conductive Ink-Coated Paper-Based Supersandwich DNA Biosensor for Ultrasensitive Detection of Neisseria gonorrhoeae

Biosensors (Basel). 2023 Apr 18;13(4):486. doi: 10.3390/bios13040486.

Authors

Niharika Gupta¹, D Kumar², Asmita Das¹, Seema Sood³, Bansi D Malhotra¹

Affiliations

¹ Department of Biotechnology, Delhi Technological University, Delhi 110042, India.
² Department of Applied Chemistry, Delhi Technological University, Delhi 110042, India.
³ Department of Microbiology, All India Institute of Medical Sciences, Ansari Nagar, New Delhi 110016, India.

Abstract

Herein, we report results of the studies relating to the development of an impedimetric, magnetic bead-assisted supersandwich DNA hybridization assay for ultrasensitive detection of Neisseria gonorrhoeae, the causative agent of a sexually transmitted infection (STI), gonorrhea. First, a conductive ink was formulated by homogenously dispersing carboxylated multiwalled carbon nanotubes (cMWCNTs) in a stable emulsion of terpineol and an aqueous suspension of carboxymethyl cellulose (CMC). The ink, labeled C5, was coated onto paper substrates to fabricate C5@paper conductive electrodes. Thereafter, a magnetic bead (MB)-assisted supersandwich DNA hybridization assay was optimized against the porA pseudogene of N. gonorrhoeae. For this purpose, a pair of specific 5' aminated capture probes (SCP) and supersandwich detector probes (SDP) was designed, which allowed the enrichment of target gonorrheal DNA sequence from a milieu of substances. The SD probe was designed such that instead of 1:1 binding, it allowed the binding of more than one T strand, leading to a 'ladder-like' DNA supersandwich structure. The MB-assisted supersandwich assay was integrated into the C5@paper electrodes for electrochemical analysis. The C5@paper electrodes were found to be highly conductive by a four-probe conductivity method (maximum conductivity of 10.1 S·cm^-1). Further, the biosensing assay displayed a wide linear range of 100 aM-100 nM (10⁹ orders of magnitude) with an excellent sensitivity of 22.6 kΩ·(log[concentration])^-1. The clinical applicability of the biosensing assay was assessed by detecting genomic DNA extracted from N. gonorrhoeae in the presence of DNA from different non-gonorrheal bacterial species. In conclusion, this study demonstrates a highly sensitive, cost-effective, and label-free paper-based device for STI diagnostics. The ink formulation prepared for the study was found to be highly thixotropic, which indicates that the paper electrodes can be screen-printed in a reproducible and scalable manner.

Keywords: DNA biosensor; Neisseria gonorrhoeae; conductive ink; electrochemical; infectious diseases.

MeSH terms

Biosensing Techniques* / methods
DNA / analysis
Electrochemical Techniques / methods
Electrodes
Gonorrhea* / diagnosis
Humans
Ink
Nanotubes, Carbon* / chemistry
Neisseria gonorrhoeae / genetics

Substances

Nanotubes, Carbon
DNA

Grants and funding

This research received no external funding.