Detection Of Small Molecules Research Articles

Development of a specific biosensor for sesquiterpene based on SELEX and directed evolution platforms

Biosensors are essential in synthetic biology, particularly for detecting compounds without distinct visual markers. In this study, a riboswitch biosensor specifically responsive to a sesquiterpene — amorpha-4,11-diene was developed for the first time. Through SELEX-SMB, a high-affinity aptamer library comprising 81,520 sequences was generated. Subsequent screening via the TBG-directed evolution platform identified riboswitch5, which downregulates downstream gene expression in response to amorpha-4,11-diene within a concentration range of 10–100 mg/L, achieving a log₂Foldchange of −0.51 at 100 mg/L. Structural predictions, combined with molecular docking and molecular dynamics simulations, revealed that ligand binding induced conformational changes that stabilize the riboswitch and enhance its repressive effect. This biosensor represents a powerful tool for the detection and regulation of small molecules, with broad applications in strain engineering, pathway regulation, and metabolic optimization in synthetic biology.

Microfluidic Optical Aptasensor for Small Molecules Based on Analyte-Tuned Growth of Gold Nanoseeds and Machine Learning-Enhanced Spectrum Analysis: Rapid Detection of Mycotoxins.

Natural toxins, mainly small molecules, are a category of chemical hazards in agri-food systems that pose threats to both public health and food security. Current standard methods for monitoring these toxins, predominantly based on liquid chromatography-mass spectrometry, are costly, labor-intensive, and complex. This study presents the development of a novel microfluidic optical aptasensor for rapid detection of small molecules based on analyte-tuned growth of gold nanoseeds combined with machine learning-enhanced spectrum analysis. We discovered and optimized a previously unreported growth pattern of aptamer-coated nanoparticles in the presence of different concentrations of analyte, enabling the detection of a major mycotoxin in food. The entire analysis was miniaturized on a customized microfluidic platform, allowing for automated spectral acquisition with precise liquid manipulation. A machine learning model, based on random forest with feature engineering, was developed and evaluated for spectrum analysis, significantly enhancing the prediction of mycotoxin concentrations. This approach extended the detection limit determined by the conventional method (∼72 ppb with high variation) to a wider range of 10 ppb to 100 ppm with high accuracy (overall mean absolute percentage error of 5.7%). The developed analytical tool provides a promising solution for detecting small molecules and monitoring chemical hazards in agri-food systems and the environment.

CRISPR-Cas14a and allosteric transcription factors empowered cell-free electrochemical biosensor for highly sensitive and stable detection of progesterone in multiple scenarios

In this study, a cell-free electrochemical assay based on allosteric transcription factors (aTFs) and CRISPR-Cas14a was developed for the detection of progesterone in trace samples. This electrochemical biosensor helps to overcome the drawbacks of the traditional fluorescence assay based on the CRISPR-Cas system and aTFs combined for non-nucleic acid targets that is poorly effective for the detection of colored samples. By comparing and optimizing the concentration and length of the probes in the straight chain and hairpin structure, the sensor performance was improved. In addition, different sgRNA from other studies was designed to overcome the effect of sequence folding in the space region on Cas14a activation. Based on these optimization results, we constructed an electrochemical sensor for progesterone quantification in the range of 66.7pM to 3.33 × 10−1μM. This method requires only 2 μL of sample and does not necessitate complex pretreatment steps, with detection completed within 1.5 h. The method has been successfully applied to food, environmental, and biological samples, with recovery rates between 82.65% and 109%. This suggests that CRISPR and allosteric transcription factor-powered electrochemical detection methods have significant potential for use in the field of small molecule detection under various scenarios.

Sample-to-answer point-of-care testing platform for quantitative detection of small molecules in blood using a smartphone-and microfluidic-based nanoplasmonic biosensor

Rapid and ultrasensitive detection of small molecules is of great significance in point-of-care testing (POCT) for health management and biomarker detection for many diseases. In this study, a novel POCT device integrated with a metasurface plasmon resonance (MetaSPR) chip with diffusion film enhancement was designed and controlled by a smartphone app for small molecule ultrasensitive detection based on competitive immunoassays. In this method, the sample competes with the antigen immobilized on the chip, and labeled antibodies are pumped from the sample pad to the chip for multiplexed and sample-to-answer analysis of blood. The entire process of detection is 14.3-fold less time (210 s in total) compared to an enzyme-linked immunosorbent assay (ELISA). For this assay, the limit of detection was 0.36 ppt and 1.05 ppt for testosterone and 25-hydroxyvitamin D3, respectively, and showed 100- to 1000-fold greater sensitivity than that of ELISA. Collectively, the data suggest that this novel POCT platform, based on a MetaSPR biosensor, provides ultrasensitive, rapid, regenerable, versatile, and real-time quantitative analysis for applications in health management and early diagnosis.

Novel multi-layered MXene as laser desorption ionization mass spectrometry matrix for rapid detection of small biomolecules

Five two-dimensional multi-layered MXenes namely MO2C, Ti2C, V2C, Nb2C and Ta2C were synthesized by a one-step exfoliation process and evaluated as laser desorption ionization mass spectrometry (LDI-MS) matrices for fast detection of small biomolecules for the first time. Among them, Nb2C exhibited the best performance due to its high carrier mobility, strong photo-induced charge transfer resonance and good UV absorption ability. A simple, universal and efficient LDI-MS platform was established for small biomolecule analysis based on the Nb2C matrix. Key factors that affect LDI-MS efficacy were investigated and the established method demonstrated ultraclean MS background, high sensitivity with detection limits low to fmol level and good reproducibility. The Nb2C-based LDI-MS platform was successfully applied in the rapid determination of 13 amino acids (AAs) in human serum and 3 AAs, as potential biomarkers, were found to be significantly down-regulated in major depressive disorder patients. This work proved the feasibility of Nb2C MXene as an LDI-MS matrix and provided a promising and universal platform for the rapid detection of small molecules in biomedical research.

Metabolomics of Chinese Hamster Ovary Cells.

Increasing demand of protein biotherapeutics produced using Chinese hamster ovary (CHO) cell lines necessitates improvement in the production yield of the bioprocess. Various cell engineering, improved media formulation and process-designbased approaches utilizing the power of OMICS technologies, specifically, genomics and proteomics, have been employed; however, the potential of metabolomics largely remains unexplored. Metabolomics enables the detection, identification, and/or quantitation of small molecules, commonly known as metabolites, in and around the cells and may help to unlock the cellular molecular mechanism(s) that regulates cell growth and productivity in the bioprocess and improves cellular performance during the bioprocess. Currently, liquid chromatography (LC)/gas chromatography (CG)- coupled with mass-spectrometry (MS) and nuclear magnetic resonance (NMR) spectroscopy are the most commonly used approaches for metabolomics. Therefore, in this chapter, we have discussed the standard procedures of investigating CHO metabolites using LC/GC-MS and/or NMR-based approaches.

Identification of new SdiA regulon members of Escherichia coli, Enterobacter cloacae, and Salmonella enterica serovars Typhimurium and Typhi.

Many bacterial species detect their own population density through the production, release, and detection of small molecules (quorum sensing). Salmonella and other Enterobacteriaceae have a modified system that detects the N-acyl-homoserine lactones of other bacteria through the solo quorum sensing receptor SdiA, a behavior known as eavesdropping. The roles of sdiA-dependent eavesdropping in the lifecycles of these bacteria are unknown. In this study, we identify sdiA-dependent transcriptional responses in two clinically relevant serovars of Salmonella, Typhimurium and Typhi, and note that their responses are partially conserved. We also demonstrate for the first time that sdiA-dependent regulation of genes is partially conserved in Enterobacter cloacae and Escherichia coli as well, indicating a degree of commonality in eavesdropping among the Enterobacteriaceae.

Effective Identification and Highly Sensitive Quantification of Fructo-oligosaccharide Isomers with Bi2Se3 Nanosheet-Assisted Laser Desorption Ionization Mass Spectrometry.

The growing interest in fructo-oligosaccharides (FOSs) necessitates the effective monitoring of product quality. Identifying and quantifying FOS isomers from the same sources are challenging. Here, we report a new method using Bi2Se3 nanosheets as the matrix for matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS), achieving effective differentiation of oligosaccharide isomers through MALDI-MS/MS. Notably, four isomers of pentasaccharides and two isomers of heptasaccharides were successfully identified, with a remarkably low limit of detection of 0.06 pmol. Our approach enabled the specific quantification of 1F-fructofuranosylnystose in commercial FOS products, positioning it as a promising tool for oligosaccharide isomer quantification in nutritional food products. Furthermore, this technique facilitates the rapid and sensitive detection of various saccharides and a wide range of other small molecules with enhanced signal intensities and improved reproducibility. Overall, it facilitates the rapid, selective, and sensitive detection of various saccharides and other small molecules, enhancing analytical chemistry and food science applications.

Preparation of a V–COF@SWCNTs-COOH/SPCE supported molecularly imprinted electrochemical sensor for real-time detection of trace sulfadimidine

To overcome the limitations of insufficient sensitivity and poor specificity of portable screen-printed carbon electrode-electrochemical sensors (SPCE-EC) in practical applications, we prepared carrier composites of carboxylic single-walled carbon nanotubes vertically grafted by covalent organic frameworks (v-COF@SWCNTs-COOH) and coated with a molecularly imprinted polymer (MIP) of sulfadimidine (SM2). 55 °C hot steam elution is more eco-friendly than traditional organic solvent elution. The results showed that when the mass ratio of DBA to DBA-SWCNTs was 1:1, the v-COF@SWCNTs-COOH obtained by the two-step synthesis method could increase the electrical signal up to 2.33-fold of the bare electrode. The bifunctional monomer MIP prepared on the above structure enhanced the signal response by 2.91-fold, with a high imprint factor of 20. The assembled MIP/v-COF@SWCNTs-COOH/SPCE were analyzed by differential pulse voltammetry (DPV) with a high sensitivity of 0.21 nM for LOD and 0.70 nM for LOQ. In milk and fish samples, the recovery rate was 95.0 %–104.8 %. The validation of authentic pork samples with the statutory LC-MS/MS method showed no significant difference (P > 0.05). The sensor's performance indicators remained robust after five repeated uses. Therefore, the MIP/v-COF@SWCNTs-COOH/SPCE combines the cheapness and portability of SPCE, while the sensitivity and specificity of small molecule detection were significantly improved.

Advancements and Applications of Single-Atom Nanozymes in Sensing Analysis

Single-atom nanozymes, with their atomically dispersed metal active sites, distinctive atom utilization rate, and tunable electronic structure, demonstrate great promise in the field of sensing analysis. This paper reviews the latest research progress on single-atom nanozymes in sensing applications. We classify single-atom nanozymes based on both their structural characteristics, such as carbon-based carriers, frameworks and their derivatives, metal oxides, metal sulfides, and organic polymer carriers, and their unique catalytic properties, including peroxidase, oxidase, catalase, superoxide dismutase, and multi-enzyme mimetic activities. Furthermore, we discuss the application of single-atom nanozymes in the sensitive detection of biological small molecules, antioxidants, ions, enzyme activities and their inhibitors, as well as cells and viruses. Finally, we highlight the opportunities and challenges for advancing the practical application and further research of single-atom nanozymes in the field of sensing analysis.

B-154 Development and Evaluation of an Automated High-Throughput Magnetic Bead-Based pre-treatment Platform for Lipid-Soluble Vitamins in Clinical Mass Spectrometry

Abstract Background Mass spectrometry has become a critical tool in clinical diagnostics due to its unparalleled sensitivity, specificity, and ability for multiplex analysis, redefining standards in small molecule detection. However, its efficacy is limited by complex sample preparation requirements, such as purification and enrichment. Traditional pre-treatment methods often involve labor-intensive, error-prone procedures that can dilute samples, thereby necessitating enhanced sensitivity in mass spectrometry. Addressing these challenges, we developed an automated, high-throughput pre-treatment platform using magnetic bead technology to streamline the preparation of small molecules for analysis via liquid chromatography-tandem mass spectrometry (LC-MS/MS). This study assesses the platform's effectiveness and reliability using lipid-soluble vitamins as a test case. Methods We developed a magnetic bead-based extraction process for lipid-soluble vitamins (Vitamins A, 25-hydroxyvitamin D2, 25-hydroxyvitamin D3, E, and K1) from clinical serum samples, utilizing Zybio's EXM3000 for automation. Protein-bound vitamins were dissociated using methanol and isopropanol, followed by magnetic bead extraction. The extracted vitamins underwent LC-MS/MS analysis. Comparative analysis with traditional liquid-liquid extraction methods, using 20 clinical samples and commercially available kits, evaluated this novel approach's analytical efficacy. Results The method showed excellent linearity (r > 0.99) for the target compounds. The imprecision ranged from 1.5% to 9.3%, and the accuracy (recovery rate) was between 91.5% and 113.4%. The limit of quantification, repeated six times, had a deviation of -8.5% to 13% and a CV of 1.5%-6.4%, meeting industry standards and EP document requirements. Comparisons with commercial kits in 20 clinical samples showed deviations within ±15%, and the 95% confidence interval for expected bias met the testing requirements. The method also demonstrated high correlation with the commercial kits, as evidenced by regression equations for Vitamin A: y = 0.9732x - 1.7985, 25-hydroxyvitamin D2: y = 1.0163x - 0.0055, 25-hydroxyvitamin D3: y = 1.0597x - 0.7968, Vitamin E: y = 1.0569x - 0.2917, and Vitamin K1: y = 1.0055x - 0.0039. Conclusions This study showed excellent performance of a novel automated high-throughput magnetic bead sample preparation platform for small molecules in clinical mass spectrometry. This technology overcomes the complexities associated with sample pre-treatment in clinical mass spectrometry and holds broad clinical application prospects in mass spectrometric analysis.

Fluorescence Label‐Free Doxorubicin Sensor Using Polystyrene Sulfonate as a Synthetic Receptor in Whispering Gallery Mode Microresonators

AbstractThe conventional quantification of Doxorubicin (DXR), a crucial anticancer drug, typically relies on complex and expensive techniques, limiting measurements beyond hospital settings and at different time intervals. Here, a label‐free fluorescence sensor is presented for DXR utilizing Whispering Gallery Mode (WGM) resonators. The resonators are based on fluorescent microparticles coated with a nanometer‐thick polystyrene sulfonate (PSS) layer serving as a synthetic receptor. The sensor demonstrates linear response for both position and width of the resonance peaks within the concentration range of 1–10 µg mL−1 of DXR of clinical interest. It also exhibits good selectivity against other chemotherapy drugs and can operate in complex biological fluids. The findings underscore the potential of WGM resonators as versatile multiparameter sensors for the sensitive and selective detection of small molecules, also in complex biological media.

Development of a CRISPR/Cas12a-facilitated fluorescent aptasensor for sensitive detection of small molecules

The integration of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated proteins (Cas) exhibits superior performance in biosensor construction. And the distinctive role of aptamers in target recognition has long been a focal point of research. Through the combination of Cas12a with cis-cleavage activity and aptamer with specific recognition, a simple and rapid fluorescent biosensor has been constructed. Interestingly, with modified fluorescent and quenching groups at two ends, aptamers play a dual role: primarily as the elements for target recognition and additionally functioning act as the fluorescent probe for signal output. Coupling with cis-cleavage of Cas12a, the demand of additional signal probes is eliminated, thus simplifying the reaction system and enhancing result accuracy. Taking okadaic acid (OA) as a representative small molecule model to evaluate the sensor's performance, a simple and straightforward detection method was established. Following this, the universality of the constructed fluorescent aptasensor was validated by incorporating an adenosine triphosphate (ATP) aptamer. Consequently, the CRISPR/Cas12a-assisted aptasensor was demonstrated to serve as a versatile detection platform for small molecules in food safety and clinical diagnostics. In the forthcoming research endeavors, it can be further extended for applications in environmental analysis and various other fields.

Development of a carboxymethyl chitosan functionalized slide for small molecule detection using oblique-incidence reflectivity difference technology.

Label-free optical biosensors have become powerful tools in the study of biomolecular interactions without the need for labels. High throughput and low detection limit are desirable for rapid and accurate biomolecule detection. The oblique-incidence reflectivity difference (OI-RD) technique is capable of detecting thousands of biomolecular interactions in a high-throughput mode, specifically for biomolecules larger than 1000 Da. In order to enhance the detection capability of OI-RD for small molecules (typically < 500 Da), we have developed a three-dimensional biochip that utilized carboxymethyl chitosan (CMCS) functionalized slides. By investigating various factors such as sonication time, protein immobilization time, CMCS molecular weight, and glutaraldehyde (GA) functionalization time, we have achieved a detection limit of 6.8 pM for avidin (68 kDa). Furthermore, accurate detection of D-biotin with a molecular weight of 244 Da has also been achieved. This paper presents an effective solution for achieving both high throughput and low detection limits using the OI-RD technique in the field of biomolecular interaction detection.

Digital One-Step Competitive Detection of a Small Molecule in Synthetic and Environmental Waters.

Optical methods for single-molecule analysis hold the promise of accurate, sensitive, and rapid detection of target molecules. Here, we demonstrate the efficiency of such an approach for the competitive detection of small molecules in water. Our biosensing method is based on a combination of a single-DNA biochip for the parallelization of tethered particle motion real-time measurements with antibodies and modified targets as molecular competitors. The antibodies are coupled to the particles tethered to the surface by a long DNA bearing in its middle the molecular competitor bound to the antibodies. Competitive target binding leads to a detectable conformational change of the DNA tethers from looped to unlooped in proportions related to the target concentration. We thus managed to detect fluorescein, chosen as a model of a target molecule, in freshwater of various qualities, from solutions prepared with ultrapure water to more complex matrices such as river water and wastewater treatment plant effluent samples. Similar dose-response curves were obtained under these various conditions in a wide range of concentrations from nanomolar to micromolar with a limit of detection around 2 nM.

“Partner” cellulose gel with “dialysis” function: Achieve the integration of filtration-enrichment-SERS detection

Hydrogel and aerogel materials have garnered significant attention in constructing effective surface-enhanced Raman spectroscopy (SERS) substrates due to their excellent adsorption capabilities, high specific surface area, and abundant chemical groups. However, in liquids with complex compositions, non-specific adsorption of macromolecules can lead to surface scaling and pore clogging of the substrate material, limiting the selective enrichment and SERS detection of target molecules. To address this, an innovative aerogel-chimeric hydrogel material (CH@S-CNF/SA/Ag NPs) was developed. The aerogel component, with its high specific surface area and electronegative properties, functions as a SERS “chip” for adsorption and detection of target molecules. Simultaneously, the mesoporous structure of the hydrogel “shell” effectively filters macromolecules from the solution. These CH@S-CNF/SA/Ag NPs were utilized as SERS substrate materials for detecting urine from healthy individuals and patients with chronic kidney disease stage 5 (CKD5). When combined with machine learning algorithms, the detection accuracy reached 99.50%. This work represents a significant advancement in the specific adsorption and SERS detection of small molecules in complex biological samples such as urine and blood.

Recent Application of Nanopores in the Detection of Small Organic Molecules

Recent Application of Nanopores in the Detection of Small Organic Molecules

Paper chip-based colorimetric biosensor for glucose by using CuFe2O4@GO as nanozyme

The development of nanomaterials with notable enzyme-like activity is of great significance to the field of bioanalysis. Herein, a nanomaterial composed of copper ferrite nanoparticles (CuFe2O4 NPs) and graphene oxide (GO) was successfully prepared (CuFe2O4@GO). The obtained CuFe2O4@GO was proved to have peroxidase-like (POD-like) activity, which could catalyze the oxidation of 3,3′,5,5′-tetramethylbenzidine (TMB) to generate oxidized TMB (ox-TMB) in the presence of H2O2 with an obvious color change. On this basis, a colorimetric biosensor using a UV–vis spectrometer as readout and a paper chip-based colorimetric biosensor using a smartphone as readout was constructed for glucose detection, respectively, as glucose oxidase (GOD) could oxidize glucose to form H2O2. The UV–vis spectrometer-based approach exhibits a linear range of 0 to 1000 μM and a limit of detection (LOD) of 0.79 μM for glucose. The smartphone-based method shows a linear range of 0.1 to 50 mM and a LOD of 0.04 mM for glucose. Moreover, the fabricated colorimetric biosensors have good selectivity towards glucose and the sample matrixes cannot affect the detection of glucose. Finally, the concentration of glucose in human serum samples was successfully detected using two different ways, and the results are close to the clinical values. The proposed method embraces several good merits. Firstly, the colorimetric biosensors combining UV–vis spectrometer and smartphone offer precise results, which could effectively prevent the occurrence of false events. Secondly, paper-based microfluidic analytical device (μPAD) as analytical platform and smartphone as readout provides a versatile approach for constructing sensitive, cost-effective, and simple colorimetric biosensor for the visual monitoring of glucose. Lastly, except for glucose detection, this work also shows great application prospects for other small molecules detection that could generate H2O2.

Self-Assembled Monolayer Transporters Enable Reagentless Analysis of Small Molecule Analytes.

The detection of small molecules beyond glucose remains an ongoing challenge in the field of biomolecular sensing owing to their small size, diverse structures, and lack of alternative non-enzymatic sensing methods. Here, we present a new reagentless electrochemical approach for small molecule detection that involves directed movement of electroactive analytes through a self-assembled monolayer to an electrode surface. Using this method, we demonstrate detection of several physiologically relevant small molecules as well as the capacity for the system to operate in several biological fluids. We anticipate that this mechanism will further improve our capacity for small molecule measurement and provide a new generalizable monolayer-based technique for electrochemical assessment of various electroactive analytes.

Versatile and efficient fabrication of signal “turn‐on” lateral flow assay for ultrasensitive naked eye detection of small molecules based on self‐assembled fluorescent gold nanoclusters‐antigen aggregates

Versatile and efficient fabrication of signal “turn‐on” lateral flow assay for ultrasensitive naked eye detection of small molecules based on self‐assembled fluorescent gold nanoclusters‐antigen aggregates