Molecular recognition elements (MREs) can be short sequences of single-stranded DNA, RNA, small peptides, or antibody fragments. They can bind to user-defined targets with high affinity and specificity. There has been an increasing interest in the identification and application of nucleic acid molecular recognition elements, commonly known as aptamers, since they were first described in 1990 by the Gold and Szostak laboratories. A large number of target specific nucleic acids MREs and their applications are currently in the literature. This review first describes the general methodologies used in identifying single-stranded DNA (ssDNA) aptamers. It then summarizes advancements in the identification and biosensing application of ssDNA aptamers specific for bacteria, viruses, their associated molecules, and selected chemical toxins. Lastly, an overview of the basic principles of ssDNA aptamer-based biosensors is discussed.

Widespread use of the chlorotriazine herbicide, atrazine, has led to serious environmental and human health consequences. Current methods of detecting atrazine contamination are neither rapid nor cost-effective. In this work, atrazine-specific single-stranded DNA (ssDNA) molecular recognition elements (MRE) were isolated. We utilized a stringent Systematic Evolution of Ligands by Exponential Enrichment (SELEX) methodology that placed the greatest emphasis on what the MRE should not bind to. After twelve rounds of SELEX, an atrazine-specific MRE with high affinity was obtained. The equilibrium dissociation constant (K_d) of the ssDNA sequence is 0.62 ± 0.21 nM. It also has significant selectivity for atrazine over atrazine metabolites and other pesticides found in environmentally similar locations and concentrations. Furthermore, we have detected environmentally relevant atrazine concentrations in river water using this MRE. The strong affinity and selectivity of the selected atrazine-specific ssDNA validated the stringent SELEX methodology and identified a MRE that will be useful for rapid atrazine detection in environmental samples.

Alpha toxin is one of the major virulence factors secreted by Staphylococcus aureus, a bacterium that is responsible for a wide variety of infections in both community and hospital settings. Due to the prevalence of S. aureus related infections and the emergence of methicillin-resistant S. aureus, rapid and accurate diagnosis of S. aureus infections is crucial in benefiting patient health outcomes. In this study, a rigorous Systematic Evolution of Ligands by Exponential Enrichment (SELEX) variant previously developed by our laboratory was utilized to select a single-stranded DNA molecular recognition element (MRE) targeting alpha toxin with high affinity and specificity. At the end of the 12-round selection, the selected MRE had an equilibrium dissociation constant (K_d) of 93.7 ± 7.0 nM. Additionally, a modified sandwich enzyme-linked immunosorbent assay (ELISA) was developed by using the selected ssDNA MRE as the toxin-capturing element and a sensitive detection of 200 nM alpha toxin in undiluted human serum samples was achieved.

The unique properties of single-wall carbon nanotubes (SWCNT) make them useful in many new technologies and applications. The interaction of DNA and SWCNT is of interest for many uses, including molecular sensors. This study determined polymerase chain reaction (PCR) efficiency in amplifying a 76 base pair DNA sequence in the presence of SWCNT, of heterogeneous “Mix” and (6,5)-enriched chiralities, associated with three DNA sequences. The dependence of PCR efficiency on the concentration of DNA:SWCNT preparations was measured, as well as their age and level of dispersion (less than one month or between four and ten months). Additionally, the ability to directly amplify the DNA sequence associated with the SWCNT scaffold was investigated. In PCRs with DNA:SWCNT preparations less than one month old, concentrations greater than or equal to 0.1 mg/mL inhibited the PCR reaction. In PCRs with older preparations, no inhibition was seen at 0.01 or 0.1 mg/mL, with amplification at 1 mg/mL in some samples. Additionally, our studies showed that the DNA directly associated with the SWCNT can be amplified using PCR. This work provides an inhibitory concentration of DNA-dispersed SWCNT in PCR reactions for different preparations as well as a basis for future DNA:SWCNT studies that require PCR amplification. This will be useful for future studies focused on the use of SWCNT in molecular sensing technologies.

Bromacil is a widely used herbicide that is known to contaminate environmental systems. Due to the hazards it presents and inefficient detection methods, it is necessary to create a rapid and efficient sensing device. Towards this end, we have utilized a stringentin vitroselection method to identify single-stranded DNA molecular recognition elements (MRE) specific for bromacil. We have identified one MRE with high affinity (Kd=9.6 nM) and specificity for bromacil compared to negative targets of selection and other pesticides. The selected ssDNA MRE will be useful as the sensing element in a field-deployable bromacil detection device.

Many large-scale applications of the organophosphate pesticide malathion have led to widespread environmental contamination. Concentrations are found in the environment well above those which are harmful to humans and environmental organisms. No current method of detection for this pesticide is rapid, cost-effective, and specific for malathion. Therefore, we utilized a stringent Systematic Evolution of Ligands by Exponential Enrichment (SELEX) process to identify a Molecular Recognition Element (MRE) for malathion. This MRE was identified from a large ssDNA library and has an equilibrium dissociation constant (K_d) in the low-nanomolar range. Additionally, it has significant selectivity for malathion in comparison to various other pesticides and metabolites of malathion, which were used as negative targets of selection. The high affinity and selectivity of the ssDNA MRE for malathion is a product of the stringent SELEX selection scheme and will be useful for rapid, inexpensive, and specific detection of malathion in the environment.