Cooperative Split DNA Activation of CRISPR-Cas12a for Sensitive and Selective Detection of Flunixin

The increasing importance of monitoring environmental changes induced by chemical stimuli affects various aspects of human life, including daily routines, healthcare, and manufacturing. [1] Addressing this demand necessitates advanced sensor units with specific attributes, such as sensitivity, excellent selectivity, rapid response times, stability, and reusability. [2] Conducting polymers (CPs) have arisen as a promising avenue in the field of sensors due to their advantages, including ease of fabrication, cost-effectiveness, lightweight, and the potential to tailor surface functional groups. [3] However, they face challenges in sensitively detecting target molecules due to their relatively weak interaction, and achieving selectivity is a key objective. In light of these challenges, we conducted research on molecularly imprinted polymer (MIP)-based CP sensors to achieve sensitive and selective detection of target analytes. [4] MIPs are functional porous materials with high-affinity binding sites that closely match the dimension and functionality of the analyte. [5] By establishing active sensing sites through covalent or non-covalent bonding between the sensing material and target molecules, MIPs can mimic biological antibodies. MIPs offer various advantages compared to antibodies, including ease of production, cost-effectiveness, reusability, chemical stability, and long-term storage at room temperature. [6]While research on MIPs is actively conducted across various applications such as chemical and biosensors, absorbents, membranes, and catalysts, the use of MIPs in electroanalytical methods still presents challenges such as low electrical conductivity, difficulty in immobilizing MIPs on electrode surfaces, and limited accessibility to binding sites. These limitations can be resolved by employing conducting polymers (CPs) to create MIPs. Molecularly imprinted conducting polymers (MICPs) combine the selectivity of MIPs with the intrinsic conductivity and processability of CPs, resulting in sensors with improved signal transduction, uniformity of binding sites, and simplified fabrication and immobilization. [7] Recently, MICP-based electrochemical sensors have gained significant attention due to their advantages, including simplified fabrication and immobilization, intrinsic electrical conductivity, and uniform binding sites. [8] However, MICPs also present challenges, such as maintaining structural integrity during template removal, limited availability of functional groups in conducting monomers, and lower reactivity toward target molecules.In this study, we demonstrated the rational design and development of MICP-based sensors tailored for the selective and sensitive detection of target analytes such as furaneol and cortisol. [9] By systematically comparing insulating and conducting MIPs, we revealed the crucial role of functional monomer structure and hydrogen bonding in enhancing imprinting efficiency and sensor performance. The integration of conducting polymers not only enabled direct signal transduction in electrochemical and chemiresistive formats but also allowed for scalable and reproducible fabrication. [10] In addition, we introduced a comparative analysis of different conducting monomers, highlighting the benefits of NH-rich structures for stress biomarker detection. These findings demonstrate that the rational design of conducting polymers combined with MIPs enables the development of low-cost, scalable, and high-performance sensing platforms for real-world applications in food monitoring and healthcare.

Prostate specific antigen (PSA) is a widely used marker for the diagnosis of prostate cancer, and the increasing attention has been attracted on the development of rapid assay using biosensing technology. However, it remains challenging for the sensitive and selective detection of PSA in clinical samples. Here, we report a label-free microfluidic paper-based analytical device for highly sensitive electrochemical detection of PSA. The paper device was fabricated with wax printing to generate hydrophobic and hydrophilic layers for the construction of microfluidic channel, followed by screen-printing of three electrodes including working, counter and reference electrode. Gold nanoparticles (AuNPs)/reduced graphene oxide (rGO)/thionine (THI) nano composites were synthesized and characterized, which were coated onto working electrodes for the immobilization of DNA aptamer probe. THI servers as the electrochemical mediator to transduce the biological recognition between DNA aptamer and PSA, and the excellent conductivity of AuNPs and rGO also play a significant role of electron transfer, leading to a sensitive detection for PSA, able to detect PSA as low as 10 pg mL−1, with a linear range from 0.05 to 200 ng mL−1. We demonstrated that our electrochemical sensor for the detection of clinical serum samples, indicating that our sensor would provide a new platform for low cost, sensitive and point-of-care diagnosis of prostate cancer.

Purely electronic diagnostic devices that are sensitive to biomolecules' intrinsic charge present a very attractive platform for point‐of‐care (PoC) applications, since they can operate under label‐free conditions. In this report, a graphene‐based electrolyte‐gated field‐effect transistor is developed as an immunosensor capable of sensitive analyte detection, even in the complex environment of physiological samples. The coimmobilization of an antibody fragment (F(ab′)2) and polyethylene glycol on the graphene surface allows for a highly sensitive and selective detection of a protein analyte, with a limit of detection in the low femtomolar range, both in high ionic strength buffer and undiluted serum. Multiparametric analysis of the device's analyte‐dependent electronic response shows that the mechanism behind this very sensitive detection cannot be explained by a commonly reported electrostatic gating effect. Rather, the observed combination of charge neutrality point shifts and asymmetric mobility changes are attributed to the modulation of scattering by charged impurities, which seem to dominate the device's transfer characteristics. Furthermore, the reproducibility of the normalized signal response obtained from several different devices shows that this graphene‐based immunosensor is capable of direct and quantitative measurements of protein analytes in untreated serum, imperative for diagnostic tools geared toward PoC applications.

A reactive chromophore developed at MIT exhibits sensitive and selective detection of surrogates for G-class nerve agents. This reporter acts by reacting with the agent to form an intermediate that goes through an internal cyclization reaction. The reaction locks the molecule into a form that provides a strong fluorescent signal. Using a fluorescent sensor platform, Nomadics has demonstrated rapid and sensitive detection of reactive simulants such as diethyl chloro-phosphate (simulant for sarin, soman, and related agents) and diethyl cyanophosphate (simulant for tabun). Since the unreacted chromophore does not fluoresce at the excitation wavelength used for the cyclized reporter, the onset of fluo-rescence can be easily detected. This fluorescence-based detection method provides very high sensitivity and could enable rapid detection at permissible exposure levels. Tests with potential interferents show that the reporter is very selective, with responses from only a few highly toxic, electrophilic chemicals such as phosgene, thionyl chloride, and strong acids such as HF, HCl, and nitric acid. Dimethyl methyl phosphonate (DMMP), a common and inactive simu-lant for other CW detectors, is not reactive enough to generate a signal. The unique selectivity to chemical reactivity means that a highly toxic and hazardous chemical is present when the reporter responds and illustrates that this sensor can provide very low false alarm rates. Current efforts focus on demonstrating the sensitivity and range of agents and toxic industrial chemicals detected with this reporter as well as developing additional fluorescent reporters for a range of chemical reactivity classes. The goal is to produce a hand-held sensor that can sensitively detect a broad range of chemical warfare agent and toxic industrial chemical threats.

Sensitive and selective detection of glycoprotein based on dual-signal and dual-recognition electrochemical sensing platform

MicroRNA-21 (miR-21) is known to act as an important biomarker for cancer, in that its up-regulation is closely related to several types of malignant tumor. Sensitive and accurate detection of miR-21 using a biosensor is highly challenging. In this study, sensitive and selective detection technology for miR-21 molecules using a quartz crystal microbalance (QCM) biosensor was developed. Sandwich hybridization between miR-21 and specially designed probes and a subsequent TiO2 photocatalytic silver enhancement reaction were the driving forces for sensitive detection with high selectivity for miR-21. Using this strategic approach under optimal conditions, the novel QCM biosensor can detect miR-21 with a LOD of 0.87 pM over the entire linear range from 0.1 pM to 10 μM, with a correlation coefficient of 0.988. In addition, the developed QCM biosensor was very effective in the quantification of miR-21 in serum samples, so the proposed miRNA detection method offers great potential for the diagnosis of early disease, such as cancer and vascular diseases, and could be an excellent alternative for biological research and clinical diagnosis.

Abstract. We have previously reported several new technologies for selective and sensitive detection of quarantine race 3 biovar 2 strains of the bacterial wilt pathogen Ralstonia solanacearum, including new highly specific PCR and LAMP primers, immunomagnetic separation (IMS) for improved isolation of the pathogen from contaminated materials, and two handheld platforms to implement the LAMP-based assay in the field. Here we report on field testing of these new technologies in a naturally infested potato field in the highlands of Guatemala and compare the results to those obtained by standard reference methods, including plating on SMSA media, use of immunostrips, and quantitative PCR. The new LAMP-based assay was demonstrated to be equivalent to quantitative PCR, as sample classifications using the respective methods were absolutely consistent when directly assaying soil and plant tissue extracts. Isolation of DNA from the extracts using IMS enabled more sensitive detection in samples that otherwise appeared negative. These results suggest that the new LAMP tools are indeed viable approaches for rapid, selective, and sensitive field-based detection of race 3 biovar 2 strains of Ralstonia solanacearum, and that IMS is a useful tool for enhancing the isolation and detection of target pathogens in the field.

Rapid, sensitive, and selective quantitative detection of pyridine dicarboxylic acid (DPA) as biomarker of anthrax spores is in great demand since anthrax spores are highly lethal to human beings and animals and also potential biological warfare agents. Herein, we prepared a ratiometric fluorescence lanthanide functionalized micelle nanoprobe by "one-pot" self-assembly, with an amphiphilic ligand containing β-diketone derivative which can "immobilize" terbium ions through the coordination interaction and a fluorophore as fluorescence reference (FR). The detection strategy was ascribed to Tb3+ ions in lanthanide functionalized micelle, which can be sensitized to emit the intrinsic luminescence upon addition of DPA due to the presence of energy transfer when DPA chromophore coordinated with Tb3+ ion. The fluorescence intensity of FR remained essentially constant, leading to ratiometric fluorescence response toward DPA. The results demonstrate that the terbium functionalized micelle was able to sensitively detect DPA with a linear relation in the range of 0 μM to 7.0 μM in aqueous solution, which also showed remarkable selectivity to DPA over other aromatic ligands. Our work paves a new way in the design of ratiometric fluorescence lanthanide functionalized micelle nanoprobes which can be promising for selective and sensitive detection of bacterial spores or biomolecules.

We have developed a method for the rapid collection and detection of leukemia cells using a novel two-nanoparticle assay with aptamers as the molecular recognition element. An aptamer sequence was selected using a cell-based SELEX strategy in our laboratory for CCRF-CEM acute leukemia cells that, when applied in this method, allows for specific recognition of the cells from complex mixtures including whole blood samples. Aptamer-modified magnetic nanoparticles were used for target cell extraction, while aptamer-modified fluorescent nanoparticles were simultaneously added for sensitive cell detection. Combining two types of nanoparticles allows for rapid, selective, and sensitive detection not possible by using either particle alone. Fluorescent nanoparticles amplify the signal intensity corresponding to a single aptamer binding event, resulting in improved sensitivity over methods using individual dye-labeled probes. In addition, aptamer-modified magnetic nanoparticles allow for rapid extraction of target cells not possible with other separation methods. Fluorescent imaging and flow cytometry were used for cellular detection to demonstrate the potential application of this method for medical diagnostics.

Probe and analogue: Double roles of thionine for aloe-emodin selective and sensitive ratiometric detection

Photocurrent-polarity-switching photoelectrochemical and electrochemical dual-mode sensing platform for the highly selective detection of trenbolone

We fabricated a gold disk microelectrode (Au DME) and developed a label-free electrochemical aptasensor for highly sensitive and selective detection of dopamine (DA) in brain slices using an anti-DA specific aptamer as the molecular recognition element and DA oxidation signal as the analytical signal. Au DME with a disk-shaped geometry and a radius in the range of 1.25 to 4 μm was fabricated by fine-tuning the size of the gold microwire inside a borosilicate capillary using laser-assisted pulling and mechanical polishing methods for easily positioning the target object. A label-free electrochemical aptasensor was fabricated by self-assembling an anti-DA-specific aptamer on the surface of Au DME with a radius of 2 μm. The obtained aptasensor directly detected DA based on the oxidation current of DA as an analytical signal, in which the recognition of DA by the anti-DA specific aptamer immobilized on Au DME enabled DA to be close to the electrode surface and facilitated the electrochemical oxidation of DA. Benefitting from Au DME with a small capacitive current and anti-DA specific aptamer with good binding affinity to DA, the label-free electrochemical aptasensor not only sensitively detected DA with a wide linear range of 0.5 to 27 μM and a low detection limit of 0.11 μM but also selectively detected DA in the presence of other interfering neurochemicals. Moreover, the label-free electrochemical aptasensor was successfully applied in the recording of the dynamic increase in DA concentration in a striatal slice from mice upon electrical stimulation. This work provides a promising strategy for the preparation of label-free electrochemical aptasensor based on microelectrodes with high spatial resolution, sensitivity and selectivity to determine neurochemical dynamics in living systems.

Profiling of a wide range of neurochemicals in human urine by very-high-performance liquid chromatography-tandem mass spectrometry combined with in situ selective derivatization

A stable and highly luminescent 3D Eu(III)-organic framework for the detection of colchicine in aqueous environment

Lipopolysaccharides (LPS), also commonly known as lipoglycans and endotoxins, can often accidentally contaminate recombinant protein therapeutics and bacteria‐derived plasmid DNA vaccines. Since LPS can induce fever, hypotension, shock, and even death, their early and sensitive detection is necessary in relevant bioassays and for parenteral drug administration and/or biotherapeutics. In this study, an optical biosensor was developed using LPS‐specific single‐stranded DNA aptamers as LPS‐selective probes. (3‐Aminopropyl) triethoxysilane and glutaraldehyde were used as linkers to immobilize the LPS high‐affinity aptamer on glass. Subsequently, each modification step was characterized by the optical responses obtained from liquid crystals using a polarized optical microscope. The sensor's ability to detect LPS was confirmed using a broad LPS detection range (5.5 pg/mL ‐ 100 ng/mL). Despite the presence of plasmid DNA, RNA, and bovine serum albumin, the aptamer sensor showed high selectivity for LPS and could be regenerated for reuse at a low pH, thus, providing a promising option for detecting LPS in complex, low pH environments.