Sensitive Detection Of Small Molecules Research Articles

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.

Non-enzymatic aptazyme-driven autonomous DNA circuits coupled with three-junction CHA for amplified live-cell ATP imaging

Live-cell imaging of adenosine triphosphate (ATP) is crucial for gaining insight into enzymatic activities and biological processes. However, it is still difficult to achieve sensitive and non-destructive ATP imaging due to the difficulty of amplifying molecules within cells and the complexity of intercellular environments. Herein, we present non-enzymatic ATP aptamer-based DNAzyme (Aptazyme)-driven autonomous DNA circuits for sensitive ATP imaging in living cells. The combination of dual amplifiers with aptazyme and catalysis hairpin assembly (CHA) circuits yielded a detection limit of 0.21 μM for ATP, which was 83.9 times more sensitive than the aptazyme-based probe without CHA circuits amplification. Moreover, the efficient carrier function and quenching effect of molybdenum disulfide (MoS2) made it possible to incorporate circuits components into cells without the requirement of transfection, thus allowing for efficient and non-destructive ATP detection within live cells. Therefore, the non-enzymatic aptazyme-driven autonomous DNA circuits present a promising novel approach for the sensitive detection of small molecules within live cells.

Sr(II) based one-dimensional coordination polymer as an efficient optical sensor for the detection of nitroaromatic explosives in aqueous medium

The detection of explosive materials holds significant importance due to their profound impact on various aspects such as environmental and biological systems, homeland security, and forensic investigations. Metal-organic frameworks (MOFs) and coordination polymers (CPs) have emerged as promising candidates for the selective and sensitive detection of small molecules. In this study, we synthesized a novel oxydiacetic acid functionalized strontium (II) coordination polymer, termed SDGA, using a cost-effective method known as the gel diffusion technique. The crystal structure, characterization, thermal stability, and photoluminescence properties of SDGA were thoroughly investigated by using single crystal X-ray diffraction, FTIR, UV–visible, TG/DTG, and fluorescence spectroscopy. The results revealed that SDGA crystallizes in a monoclinic crystal system with the P21/c space group. SDGA exhibits an emission peak at 340 nm under an excitation wavelength of 301 nm, attributed to the inter-ligand n-π* transition. Leveraging this photoluminescence behaviour, sensing experiments were conducted to detect nitrobenzene and p-nitrophenol within a concentration range of 0-10−20 M. The results indicate a decrease in fluorescence intensity with increasing concentrations of nitrobenzene and p-nitrophenol, indicative of a ‘turn-off’ mechanism.

Highly Efficient Fabrication of Fluorescent "Turn-On" Lateral Flow Strips for Highly Sensitive Detection of Small Molecules Based on Self-Assembly of AuAg Nanoclusters.

Currently, fluorescent "turn-on" lateral flow assay (FONLFA) has shown enhanced "naked eye" detection sensitivity for small molecules, while it is urgent to adopt biocompatible fluorescent nanomaterials and needs new strategies to simplify the preparation process. In this study, a highly effective method was proposed to produce FONLFA strips for the detection of small molecules. The gold-silver nanoclusters (AuAgNCs) were immobilized onto the nitrocellulose membrane of the strips by the self-assembly of poly(sodium 4-styrenesulfonate), antigen, and AuAgNCs. The immobilization process entails a straightforward mixing of the three components, taking merely 1 min, thereby bypassing the necessity for chemical modification of fluorescent nanomaterials. The strategy offers a significantly simplified process, which substantially enhances the efficiency of the strip fabrication. Utilizing this method, a FONLFA was developed for carbendazim with a visual limit of detection (vLOD) reduced by 40-fold compared with the conventional colorimetric lateral flow assay (LFA). Furthermore, the approach demonstrates versatility by enabling the immobilization of AuAgNCs and streptavidin, which facilitates the development of aptamer-based FONLFAs. The designed aptamer-based FONLFA for kanamycin exhibited a 50-fold reduction in the vLOD compared with conventional colorimetric LFAs. Therefore, FONLFA holds promising potential for widespread applications in the analysis of small molecules.

Antibody-Bridged DNAzyme Walker for Sensitive Detection of Small Molecules.

Sensitive detection of small molecules with biological and environmental interests is important for many applications, such as food safety, disease diagnosis, and environmental monitoring. Herein, we propose a highly selective antibody-bridged DNAzyme walker to sensitively detect small molecules. The antibody-bridged DNAzyme walker consists of a track, small-molecule-labeled DNAzyme walking strand, and antibody against small molecules. The track is built by co-modifying fluorophore-labeled substrates and small-molecule-labeled DNA linkers onto a gold nanoparticle (AuNP). In the absence of the target molecule, the antibody binds small molecule labels at the DNAzyme walking strand and the DNA linker, driving the DNAzyme walking strand on the surface of the AuNP. The attached DNAzyme walking strand moves along the track and cleaves substrates to generate high fluorescence signals to achieve signal amplification. As target molecules exist, they competitively bind with antibody to displace the small-molecule-labeled linker and DNAzyme walking strand, rendering the DNAzyme walker inactive in substrate cleavage and causing weak fluorescence. By using this antibody-bridged DNAzyme walker, we achieved sensitive detection of two biologically important small molecules, digoxin and folic acid. This work provides a new paradigm by combining the signal amplification strategy of a DNA walker and immunorecognition for sensitive and selective detection of small molecules.

Polymeric integration of structure-switching aptamers on transistors for histamine sensing.

Aptamers that undergo large conformational rearrangements at the surface of electrolyte-gated field-effect transistor (EG-FETs)-based biosensors can overcome the Debye length limitation in physiological high ionic strength environments. For the sensitive detection of small molecules, carbon nanotubes (CNTs) that approach the dimensions of analytes of interest are promising channel materials for EG-FETs. However, functionalization of CNTs with bioreceptors using frequently reported surface modification strategies (e.g., π-π stacking), requires highly pristine CNTs deposited through methods that are incompatible with low-cost fabrication methods and flexible substrates. In this work, we explore alternative non-covalent surface chemistry to functionalize CNTs with aptamers. We harnessed the adhesive properties of poly-D-lysine (PDL), to coat the surface of CNTs and then grafted histamine-specific DNA aptamers electrostatically in close proximity to the CNT semiconducting channel. The layer-by-layer assembly was monitored by complementary techniques such as X-ray photoelectron spectroscopy, optical waveguide lightmode spectroscopy, and fluorescence microscopy. Surface characterization confirmed histamine aptamer integration into PDL-coated CNTs and revealed ∼5-fold higher aptamer surface coverage when using CNT networks with high surface areas. Specific aptamers assembled on EG-CNTFETs enabled histamine detection in undiluted high ionic strength solutions in the concentration range of 10 nM to 100 μM. Sequence specificity was demonstrated via parallel measurements with control EG-CNTFETs functionalized with scrambled DNA. Histamine aptamer-modified EG-CNTFETs showed high selectivity vs. histidine, the closest structural analog and precursor to histamine. Taken together, these results implied that target-specific aptamer conformational changes on CNTs facilitate signal transduction, which was corroborated by circular dichroism spectroscopy. Our work suggests that layer-by-layer polymer chemistry enables integration of structure-switching aptamers into flexible EG-CNTFETs for small-molecule biosensing.

A donor-acceptor-type photoactive material based cathode molecularly imprinted photoelectrochemical sensor for luteolin

Developing new optoelectronic materials is the eternal pursuit and key strategy for building high-performance photoelectrochemical sensors. In this work, a novel organic polymer film containing D-A unit was firstly in-situ electro-synthesized on ZnS/rGO surface by using 9,9′-(9,9′-spirobi[fluorene]−2,7-diyl)-bis-9 H-carbazole (SFC) and carbazole as functional monomers. The micromorphology, element composition, structure, photoelectric efficiency and recognition ability of the key component and/or the sensing composite were investigated by SEM, XPS, XRD and transient photocurrent spectra, and the possible mechanism of charge carrier separation and transmission was proposed. Owing to the good light absorption ability, excellent film formation and imprinting ability, effective electrons-holes separation path, and satisfactory application stability, the prepared photoelectrochemical sensor exhibited good sensing performance for luteolin. Under the optimized conditions, the photocurrent intensity of the sensor showed a good linear relationship with the logarithmic concentration of luteolin from 0.001 μmol L−1 to 2 μmol L−1, with a detection limit of 0.35 nmol L−1 (S/N = 3). When applied in actual samples including carrot, peanut shells and chrysanthemum tea, highly consistent results with classical HPLC method were obtained. This work provided a new photoelectrochemical approach for the highly selective and sensitive detection of small molecules besides luteolin.

Development of CsPbBr3 fluorescence molecularly imprinted nanoprobe for highly sensitive detection of sulfapyridine

Molecularly imprinted fluorescent nanoparticles offer great potential in highly selective and sensitive detection of small molecules. Herein, a novel molecularly imprinted fluorescence nanoprobe polymer (CPB-Si-MIP) was successfully synthesized using CsPbBr3 perovskite nanocrystals (CPB PNCs) and a silane coupling agent via the ligand-assisted reprecipitation (LARP) approach, sol–gel method, and molecular imprinting technology (MIT). The results demonstrated a significant quenching of fluorescence in the CPB-Si-MIP nanoprobe with the sulfapyridine (SPD) template molecule. The linear range of SPD detection was found to be 20–100 ng/mL, with a low detection limit of 6.5 ng/mL. Furthermore, the imprinting factor (IF) of the nanoprobe was 2.50, highlighting its high specificity and recognition ability toward SPD as the target molecule.

Cell-Free Biosensing Genetic Circuit Coupled with Ribozyme Cleavage Reaction for Rapid and Sensitive Detection of Small Molecules.

Synthetic biological systems have been utilized to develop a wide range of genetic circuits and components that enhance the performance of biosensing systems. Among them, cell-free systems are emerging as important platforms for synthetic biology applications. Genetic circuits play an essential role in cell-free systems, mainly consisting of sensing modules, regulation modules, and signal output modules. Currently, fluorescent proteins and aptamers are commonly used as signal outputs. However, these signal output modes cannot simultaneously achieve faster signal output, more accurate and reliable performance, and signal amplification. Ribozyme is a highly structured and catalytic RNA molecule that can specifically recognize and cut specific substrate sequences. Here, by adopting ribozyme as the signal output, we developed a cell-free biosensing genetic circuit coupled with the ribozyme cleavage reaction, enabling rapid and sensitive detection of small molecules. More importantly, we have also successfully constructed a 3D-printed sensor array and thereby achieved high-throughput analysis of an inhibitory drug. Furthermore, our method will help expand the application range of ribozyme in the field of synthetic biology and also optimize the signal output system of cell-free biosensing, thus promoting the development of cell-free synthetic biology in biomedical research, clinical diagnosis, environmental monitoring, and food inspection.

Sensitive CRISPR-Cas12a-Assisted Immunoassay for Small Molecule Detection in Homogeneous Solution.

Sensitive detection of small molecules is crucial for many applications, like biomedical diagnosis, food safety, and environmental analysis. Here, we describe a sensitive CRISPR-Cas12a-assisted immunoassay for small molecule detection in homogeneous solution. An active DNA (acDNA) modified with a specific small molecule serves as a competitor for antibody binding and an activator of CRISPR-Cas12a. Large-sized antibody binding with this acDNA probe inactivates the collateral cleavage activity of CRISPR-Cas12a due to a steric effect. When free small molecule target exists, it replaces the small molecule-modified acDNA from antibody, triggering catalytic cleavage of DNA reporters by CRISPR-Cas12a, and strong fluorescence is generated. With this strategy, we achieved detection of three important small molecules as models, biotin, digoxin, and folic acid, at picomolar levels by using streptavidin or antibody as recognition elements. With the progress of DNA-encoded small molecules and antibody, the proposed strategy provides a powerful toolbox for detection of small molecules in wide applications.

Biomimic Nanozymes with Tunable Peroxidase-like Activity Based on the Confinement Effect of Metal-Organic Frameworks (MOFs) for Biosensing.

Biomimic nanozymes coassembled by peptides or proteins and small active molecules provide an effective strategy to design attractive nanozymes. Although some promising nanozymes have been reported, rational regulation for higher catalytic activity of biomimic nanozymes remains challenging. Hence, we proposed a novel biomimic nanozyme by encapsulating the coassembly of hemin/bovine serum albumin (BSA) in zeolite imidazolate frameworks (ZIF-8) to achieve controllable tailoring of peroxidase-like activity via the confinement effect. The assembly of Hemin@BSA was inspired by the structure of horseradish peroxidase (HRP), in which hemin served as the active cofactor surrounded by BSA as a blocking pocket to construct a favorable hydrophobic space for substrate enrichment. Benefiting from the confinement effect, ZIF-8 with a porous intracavity was identified as the ideal outer layer for Hemin@BSA to accelerate substrate transport and achieve internal circulation of peroxidase-like catalysis, significantly enhancing its peroxidase-like activity. Especially, the precise encapsulation of Hemin@BSA in ZIF-8 could also prevent it from decomposition in harsh environments by rapid crystallization around Hemin@BSA to form a protective shell. Based on the improved peroxidase-like activity of Hemin@BSA@ZIF-8, several applications were successfully performed for the sensitive detection of small molecules including H2O2, glucose, and bisphenol A (BPA). Satisfactory results highlight that using a ZIF-8 outer layer to encapsulate Hemin@BSA offers a very effective and successful strategy to improve the peroxidase-like activity and the stability of biomimic nanozymes, broadening the potential application of biocatalytic metal-organic frameworks (MOFs).

Current Challenges and Recent Developments in Mass Spectrometry-Based Metabolomics.

High-resolution mass spectrometry (MS) has advanced the study of metabolism in living systems by allowing many metabolites to be measured in a single experiment. Although improvements in mass detector sensitivity have facilitated the detection of greater numbers of analytes, compound identification strategies, feature reduction software, and data sharing have not kept up with the influx of MS data. Here, we discuss the ongoing challenges with MS-based metabolomics, including de novo metabolite identification from mass spectra, differentiation of metabolites from environmental contamination, chromatographic separation of isomers, and incomplete MS databases. Because of their popularity and sensitive detection of small molecules, this review focuses on the challenges of liquid chromatography-mass spectrometry-based methods. We then highlight important instrumentational, experimental, and computational tools that have been created to address these challenges and how they have enabled the advancement of metabolomics research.

H-FBN assisted negative ion paper spray for the sensitive detection of small molecules.

Negative ion mode paper spray mass spectrometry (PS-MS) suffers from intense background noise and unstable MS signal. For the first time, we reported fluorinated boron nitride nanosheet (h-FBN) assisted negative ion PS-MS for the detection of a series of molecules. We demonstrated that the introduction of h-FBN can greatly improve the detection sensitivity and signal stability in the negative ion mode.

Attomolar Detection of ssDNA Without Amplification and Capture of Long Target Sequences With Graphene Biosensors

Graphene-based biosensors can be produced in a scalable manner at reasonable cost, and they show significant promise for sensitive detection of small molecules and biomarkers such as proteins, single strand nucleic acids, and drug targets. Here, we describe an approach that enables a limit of detection of ~1 aM for a ssDNA target without amplification. We also show that a sensor based on a short (20mer) probe complementary to a portion of a longer (100mer) target provides enhanced sensitivity and saturation signal level. Finally, we show that graphene-based DNA biosensors can be repeatedly recycled with consistent sensor responses by melting off bound target strands. These results show the potential utility of this technology in nucleic acid detection for disease diagnostics.

A facile label-free electrochemical aptasensor constructed with nanotetrahedron and aptamer-triplex for sensitive detection of small molecule: Saxitoxin

Development of facile electrochemical aptasensors for small molecules is of great significance. Herein, the aptamer technology, DNA nanotetrahedron, DNA triplex, and electrochemistry were combined for the first time to construct a facile label-free electrochemical aptasensor for highly sensitive detection of small molecules. A typical small molecule, saxitoxin (STX, Mw. 299 g/mol), was chosen as a model target. The nanotetrahedron assisted the orientated immobilization of the aptamer- triplex on the surface of screen-printed electrodes, protecting the aptamer-triplex from absorption and entanglement and assisting the aptamer to display full accessibility to STX. Large and sensitive electrochemical signal changes were triggered along with the binding of the aptamer and STX, as the aptamer fold adaptively into specific conformational structure and caused disassembly of the triplex and dissociation of a DNA chain with Mw. exceeding 15,000 g/mol from the electrode. The developed aptasensor provided high sensitivity with a LOD of 0.92 nM and showed good practicability towards STX in real seawater samples, with well-validated recovery data (94.4%–111%), good selectivity, stability and repeatability. This study can be referred for analysis of any other small molecules.

8-Hydroxyquinolinate-Based Metal-Organic Frameworks: Synthesis, Tunable Luminescent Properties, and Highly Sensitive Detection of Small Molecules and Metal Ions.

Five new metal-organic frameworks, [Zn2L2]·2DMF·2MeOH (1), [Zn2L2(py)2] (2), [Cd2L2]·Diox·MeOH·6H2O (3), [Mn2L2]·2DMF·2MeOH (4), and [Co2L2]·2DMF·4H2O (5), were assembled by using a novel 8-hydroxyquinolinate derivative H2L with different metal ions. Complex 1 features a 3D porous network consisting of meso-helical chains ( P + M) built from metal-ligand coordination bonds. The adjacent dinuclear ZnII building blocks in 2 are connected together to generate a 2D grid network. In complex 3, each binuclear motif is bound to four ZnII ions to produce a 2D layer structure that stacks into a 3D porous structure. The framework of complex 4 is isostructural to 5, featuring a 21 helical chain built from [M2L2] units (M = Mn or Co). The adjacent meso-helices associated in parallel are interconnected by the phenolate μ2-O atoms of H2L to give rise to a 2D network. Distinct solid-state luminescence properties of 1-3 were observed, arising from their different metal nodes and frameworks. In particular, complex 1 exhibited excellent stability in both common organic solvents and H2O, thus facilitating its utility as a chemical sensor. Remarkably, luminescent 1 showed highly sensitive detection for nitroaromatic molecules in methanol and Fe3+ ion in H2O even in the presence of other interfering metal cations.

Aptamer-Structure Switch Coupled with Horseradish Peroxidase Labeling on a Microplate for the Sensitive Detection of Small Molecules.

Detection of small molecules with good sensitivity, high throughput, simplicity, and generality using aptamers is desired but still remains challenging. We described an aptamer-structure-switch assay coupled with horseradish peroxidase (HRP) labeling on microplates for sensitive absorbance and chemiluminescence detection of small molecules. This assay relies on competition for affinity binding to a limited HRP-labeled aptamer between small-molecule targets and immobilized short DNA strands complementary to the aptamer (cDNA) on a microplate. In the absence of targets, the HRP-labeled aptamer hybridizes with the cDNA on the microplate, and HRP catalyzes substrate into product, generating absorbance or chemiluminescence signals. The binding of small-molecule targets to aptamers causes displacement of HRP-labeled aptamers from the cDNA and signal decrease. In chemiluminescence-analysis mode, the assay achieved detection of aflatoxin B1 (AFB1), ochratoxin A (OTA), and adenosine triphosphate (ATP) with detection limits of 10 pM, 20 pM, and 20 nM, respectively. This assay does not require enzyme-labeled small molecules or the conjugation of small molecules on solid phase. HRP, as an enzyme label, here allows for easily obtainable and highly active signal amplification. This microplate assay is rapid and promising for high-throughput analysis. It shows potential for wide applications in the detection of small molecules.

Ag nanoparticles/ZnO nanorods for highly sensitive detection of small molecules with laser desorption/ionization mass spectrometry

In this report, a matrix-free method for high-efficiency detection of small molecules with a Ag nanoparticles/ZnO nanorods (Ag NPs/ZnO NRs) substrate as platform for surface-assisted laser desorption/ionization mass spectrometry (SALDI MS) was carried out for the first time. Ag NPs were decorated on the ZnO NRs substrate by vapor deposition. The charge separation efficiency of composite substrate was optimized by regulating the amount of evaporated Ag NPs. The excellent electron-hole separation efficiency of the Ag NPs/ZnO NRs was measured by surface photovoltage (SPV). For the same analytes, the Ag NPs/ZnO NRs substrate in negative ion mode possesses higher signal intensity and lower limit of detection (LOD). The LOD of Arginine on the Ag NPs/ZnO NRs composite substrate is only 1.0 × 10–15 M, which is 2 orders of magnitude lower than that on the ZnO NRs substrate. The other 3 amino acids (Tryptophan, Tyrosine and Lysine) could be also detected, the Ag NPs/ZnO NRs substrate showed better sensitivity and lower LODs. It indicates that the charge separation of the ZnO NRs can promote the accumulation of electrons on Ag NPs surface, which enhances laser desorption/ionization (LDI) efficiency in negative ion mode. Finally, the application of this substrate was demonstrated by analyzing the real samples, including malachite green in lake water, methadone in urine, verapamil in serum and Bradykinin 1–7 in serum, it performs highly sensitivity and linear correlation for actual samples.

Low-cost, high-performance plasmonic nanocomposites for hazardous chemical detection using surface enhanced Raman scattering

Nanoplasmonic paper holds considerable promise as an effective surface-enhanced Raman scattering (SERS) substrate for sensitive detection of small molecules. In this study, we report the development of a low cost, flexible, high-performance SERS substrate based on cellulose nanofiber/gold nanoparticle (CNF/AuNP) nanocomposites via vacuum-assisted filtration. The CNF-based matrix enables direct AuNP deposition by filtration due to its nanoscale surface roughness. In addition, the filtration process allows for precise control of the spatial distribution of AuNPs, including the number density and size ratio, which are strongly associated with interparticle plasmon coupling effects. In our experiments, the CNF-AuNP nanocomposite showed excellent SERS activity with a highly sensitive rhodamine 6 G detection limit of 10 pM and a competitive enhancement factor of 4.5 × 109. It is important to note that the control of both density number and size ratio of AuNPs contribute to enhancement of SERS. Furthermore, we demonstrated that CNF-AuNP nanocomposite-based SERS can efficiently detect two representative pesticides, thiram and tricyclazole, at trace concentrations. The limits of detection (LOD) for thiram and tricyclazole were found to be as low as 1 pM (0.3 ppt) and 10 pM (2.4 ppt), respectively, which is 100- to 1000-fold more sensitive than previously reported SERS methods. Additionally, using a CNF-AuNP nanocomposite-based swab provided sensitive detection of pesticide residues on real-world surfaces (apple peel and plant leaf), offering great potential for label-free and on-site detection of hazardous molecules.

Highly Sensitive Label-Free Detection of Small Molecules with an Optofluidic Microbubble Resonator.

The detection of small molecules has increasingly attracted the attention of researchers because of its important physiological function. In this manuscript, we propose a novel optical sensor which uses an optofluidic microbubble resonator (OFMBR) for the highly sensitive detection of small molecules. This paper demonstrates the binding of the small molecule biotin to surface-immobilized streptavidin with a detection limit reduced to 0.41 pM. Furthermore, binding specificity of four additional small molecules to surface-immobilized streptavidin is shown. A label-free OFMBR-based optical sensor has great potential in small molecule detection and drug screening because of its high sensitivity, low detection limit, and minimal sample consumption.