Characterization of Argonaute nucleases from mesophilic bacteria Paenibacillus borealis and Brevibacillus laterosporus

Thermophilic Argonaute proteins (Agos) have been shown to utilize small DNA guides for cleaving complementary DNA in vitro, which shows great potential for nucleic acid detection. In this study, we explored mesophilic Agos for the detection of small molecule by cooperating with allosteric transcription factors (aTFs). Two Agos from mesophilic bacteria, Paenibacillus borealis (PbAgo) and Brevibacillus laterosporus (BlAgo), showed nuclease activity for single-stranded DNA at moderate temperatures (37 °C) by using 5′-phosphorylated and 5′-hydroxylated DNA guides. Both Agos perform programmable cleavage of double-stranded DNA, especially in AT-rich regions of plasmid. Furthermore, we developed a simple and low-cost p-hydroxybenzoic acid detection method based on DNA-guided DNA cleavage of Agos and the allosteric effect of HosA, which expands the potential application of small molecule detection by Agos.

Anti-Metatype Antibody Screening, Sandwich Immunoassay Development, and Structural Insights for β-Lactams Based on Penicillin Binding Protein

Theoretically, sandwich immunoassay is more sensitive and has a wider working range than that of competitive format. However, it has been thought that small molecules cannot be detected by the sandwich format due to their limited size. In the present study, we proposed a novel strategy for achieving sandwich immunoassay of β-lactams with low molecular weights. Firstly, five β-lactam antibiotics were selected to bind with penicillin binding protein (PBP)2x* to form complexes. Then, monoclonal and polyclonal antibodies against PBP2x*-β-lactams complexes were produced by animal immunization. Subsequently, the optimal pairing antibodies were utilized to establish sandwich immunoassay for detection of 18 PBP2x*-β-lactam complexes. Among them, ceftriaxone could be detected at as low as 1.65 ng/mL with working range of 1–1000 ng/mL in milk. To reveal the detection mechanism, computational chemistry and molecular recognition study were carried out. The results showed that β-lactams with a large size and complex structures maybe conducive to induce conformational changes of PBP2x*, and then exhibit greater possibility of being detected by sandwich immunoassay after combination with PBP2x*. This study provides insights for subsequent investigations of anti-metatype antibody screening and sandwich immunoassay establishment for small-molecule detection.

High-sensitivity small-molecule detection of microcystin-LR cyano-toxin using a terahertz-aptamer biosensor

We demonstrate the rapid and highly sensitive detection of a small molecule, microcystin-LR (MC-LR) toxin using an aptasensor based on a terahertz (THz) emission technique named the terahertz chemical microscope (TCM).

Rational design of allosterically regulated toehold mediated strand displacement circuit for sensitive and on-site detection of small molecule metabolites

Development of small molecule biosensor enables rapid and de-centralized small molecule detection that meets the demand of routine health monitoring and rapid diagnosis. Among them, allosteric transcription factor (aTF)-based biosensors...

Using dual exonucleases to finely distinguish structural adjustment of aptamers for small-molecule detection

Dual exonucleases to finely distinguish structural adjustment of aptamers to produce absolute differentiation between digestion and inhibition.

Mass Spectrometry Imaging of Neurotransmitters

Mass spectrometry imaging (MSI) is a powerful analytical method for the simultaneous analysis of hundreds of compounds within a biological sample. Despite the broad applicability of this technique, there is a critical need for advancements in methods for small molecule detection. Some molecular classes of small molecules are more difficult than others to ionize, e.g., neurotransmitters (NTs). The chemical structure of NTs (i.e., primary, secondary, and tertiary amines) affects ionization and has been a noted difficulty in the literature. In order to achieve detection of NTs using MSI, strategies must focus on either changing the chemistry of target molecules to aid in detection or focus on new methods of ionization. Additionally, even with new strategies, the issues of delocalization, chemical background noise, and ability to achieve high throughput (HTP) must be considered. This chapter will explore previous and up-and-coming techniques for maximizing the detection of NTs.

A Simple Displacement Aptamer Assay on Resistive Pulse Sensor for Small Molecule Detection

A universal aptamer-based sensing strategy is proposed using DNA modified nanocarriers and Resistive Pulse Sensing for the rapid and label free detection of small molecules. The surface of a magnetic nanocarrier was first modified with a ssDNA aka linker which is designed to be partially complimentary in sequence to a ssDNA aptamer. The aptamer and linker form a stable dsDNA complex on the nanocarriers surface. Upon the addition of the target molecule, a conformational change takes place where the aptamer preferentially binds to the target over the linker; causing the aptamer to be released into solution. The RPS measures the change in velocity of the nanocarrier as its surface changes from dsDNA to ssDNA, and its velocity is used as a proxy for the concentration of the target. We illustrate the versatility of the assay by demonstrating the detection of the antibiotic Moxifloxacin, and chemotherapeutics Imatinib and Irinotecan.