Novel Biorecognition Elements against Pathogens in the Design of State-of-the-Art Diagnostics

Abstract

Infectious agents, especially bacteria and viruses, account for a vast number of hospitalisations and mortality worldwide. Providing effective and timely diagnostics for the multiplicity of infectious diseases is challenging. Conventional diagnostic solutions, although technologically advanced, are highly complex and often inaccessible in resource-limited settings. An alternative strategy involves convenient rapid diagnostics which can be easily administered at the point-of-care (POC) and at low cost without sacrificing reliability. Biosensors and other rapid POC diagnostic tools which require biorecognition elements to precisely identify the causative pathogen are being developed. The effectiveness of these devices is highly dependent on their biorecognition capabilities. Naturally occurring biorecognition elements include antibodies, bacteriophages and enzymes. Recently, modified molecules such as DNAzymes, peptide nucleic acids and molecules which suffer a selective screening like aptamers and peptides are gaining interest for their biorecognition capabilities and other advantages over purely natural ones, such as robustness and lower production costs. Antimicrobials with a broad-spectrum activity against pathogens, such as antibiotics, are also used in dual diagnostic and therapeutic strategies. Other successful pathogen identification strategies use chemical ligands, molecularly imprinted polymers and Clustered Regularly Interspaced Short Palindromic Repeats-associated nuclease. Herein, the latest developments regarding biorecognition elements and strategies to use them in the design of new biosensors for pathogens detection are reviewed.

Keywords: DNAzymes; antibodies; aptamers; biorecognition; biosensor; diagnosis; enzymes; pathogens; peptide nucleic acids; peptides.

Figure 1

Main components of a biosensor: analyte, biorecognition element and transducer which produces a detectable signal. The major types of biorecognition elements and analytes in the context of clinical pathogen detection are included. PNAs: peptide nucleic acids. Created with BioRender.com.

Figure 2

Three-step synthesis of monoclonal antibodies (mABs) conjugated with magnetic beads and SERS (surface-enhanced Raman spectroscopy) and fluorescence-based dual nanoprobes for the multiplex detection of Escherichia coli J5 and Francisella tularensis: (A) Magnetic bead clusters were encapsulated and conjugated to mABs to selectively capture either E. coli or F. tularensis. (B,C) For the subsequent detection step after bacterial capture, AgNP clusters were encoded with SERS reporters (red and yellow stars), stabilised by bovine serum albumin (BSA), conjugated further with fluorescent dyes and encapsulated in a polymer. (D) E. coli J5 and F. tularensis bind to the respective mABs and were magnetically separated. (E,F) Multiplex detection of sandwich immunocomplexes composed of bacteria, magnetic bead clusters and the SERS and fluorescence-based dual nanoprobes was achieved. Detection method develop by Jang et al. [43]. Created with BioRender.com.

Figure 3

Graphical representation of an antimicrobial peptide (AMP)-based biosensor used for impedimetric detection of bacteria. The AMP is immobilised on a microelectrode array. The functionalised sensor selectively captures the target cells due to the immobilised AMP. Created with BioRender.com.

Figure 4

The general steps involved in the selection of candidate aptamers towards a target bacterium by cell SELEX (systematic evolution of ligands by exponential enrichment). These include incubation of the target bacteria with a single stranded DNA (ssDNA) library, washing steps, counter-selection and PCR (polymerase chain reaction) amplification before the cycle is repeated. After all cycles are completed, candidate aptamers are sequenced. Created with BioRender.com.

Figure 5

DNAzyme biosensor proposed by Kang et al. [126]. The target(s) from lysed bacteria bind(s) to the DNAzyme sequence (orange), which undergoes a change in conformational triggering the activation of the DNAzyme. The activated DNAzyme cleaves the fluorogenic substrate at the ribonucleotide connection (R), this releases the fluorophore (F) and quencher (Q) to produce a high-fluorescence signal. Created with BioRender.com.

Figure 6

Schematic illustration of the Clustered Regularly Interspaced Short Palindromic Repeats/Cas13a (CRISPR/Cas13a) system for detection of SARS-CoV-2 ribonucleic acid (RNA) proposed by Hou et al. [148]. Reverse-Transcription Recombinase Polymerase Amplification (RT-RPA) followed by a T7 transcription is used to amplify the SARS-CoV-2 RNA. In the next step, the nuclease activity of Cas13a is activated when the guide RNA binds specifically to the open reading frame 1ab (orf1ab) gene and triggers the cleavage of the RNA reporter. The cleaved RNA reporter produces a fluorescent signal for detection of SARS-CoV-2. Created with BioRender.com.