Sensitive Detection Of Small Molecules Research Articles

Cascade Reaction-Mediated Assembly of Magnetic/Silver Nanoparticles for Amplified Magnetic Biosensing.

Conventional magnetic relaxation switching (MRS) sensor suffers from its relatively low sensitivity when it comes to the analysis of trace small molecules in complicated samples. To meet this challenge, we develop a cascade reaction-mediated magnetic relaxation switching (CR-MRS) sensor, based on the assembly of silver nanoparticles (Ag NPs) and magnetic nanoparticles (MNPs) to improve the sensitivity of conventional MRS. The cascade reaction triggered by alkaline phosphatase generates ascorbic acid, which reduces Ag+ to Ag NPs that can assemble the initially dispersed MNPs to form magnetic/silver nanoassemblies, thus modulating the state of MNPs to result in the change of transverse relaxation time. The formed magnetic/silver nanoassemblies can greatly enhance the state change of MNPs (from dispersed to aggregated) and dramatically improve the sensitivity of traditional MRS sensor, which makes this CR-MRS sensor a promising platform for highly sensitive detection of small molecules in complicated samples.

Volumetric registration of magnetic nanoparticles for optimization of quantitative immunochromatographic assays for detection of small molecules

Precise quantitative and highly sensitive detection of small molecules (haptens) is highly demanded in medicine, food quality control, in vitro diagnostics, criminalistics, environmental monitoring, etc. In the present work, the magnetic method of particle quantification and the optical methods of spectral correlation and spectral phase interferometry complement each other for optimization of a quantitative assay for measuring concentrations of small molecules. The assay employs magnetic nanoparticles as labels in rapid immunochromatographic format. The approach was demonstrated with fluorescein as a model molecule. The interferometric label-free biosensors were employed for selection of optimal reagents that produced high specificity and sensitivity. The method of magnetic particle quantification counted the magnetic labels over the entire volume of the immunochromatographic membrane to provide their distribution along the test strip. Such distribution was used for optimization of such parameters as concentrations of the used reagents and of antibody immobilized on the labels, amount of the labels and conjugates of haptens with protein carriers to realize the advanced quantitative immunochromatographic assay.

Cascaded signal amplification via target-triggered formation of aptazyme for sensitive electrochemical detection of ATP

The construction of reliable sensors for adenosine triphosphate (ATP) detection gains increasing interest because of its important roles in various enzymatic activities and biological processes. Based on a cascaded, significant signal amplification approach by the integration of the aptazymes and catalytic hairpin assembly (CHA), we have developed a sensitive electrochemical sensor for the detection of ATP. The target ATP leads to the conformational change of the aptazyme sequences and their association with the hairpin substrates to form active aptazymes, in which the hairpin substrates are cyclically cleaved by the metal ion cofactors in buffer to release the enzymatic sequences that can also bind the hairpin substrates to generate active DNAzymes. The catalytic cleavage of the hairpin substrates in the aptazymes/DNAzymes thus results in the generation of a large number of intermediate sequences. Subsequently, these intermediate sequences trigger catalytic capture of many methylene blue-tagged signal sequences on the electrode surface through CHA, producing significantly amplified current response for sensitive detection of ATP at 0.6nM. Besides, the developed sensor can discriminate ATP from analogous interference molecules and be applied to human serum samples, making the sensor a useful addition to the arena for sensitive detection of small molecules.

The characterization and validation of 17β-estradiol binding aptamers

The rapid and sensitive detection of small molecules is garnering increasing importance, and aptamers show great promise in replacing expensive, elaborate detection platforms exploiting chromatographic separation or antibody-based assays. The characterization of aptamer interaction with small molecule targets is not facile, and there is a mature need for a rapid, high-throughput technique for the analysis of aptamer-small molecule kinetics and affinity. In this work we present methodologies for the evaluation of aptamer-small molecule interactions, using the aptamers reported against the steroid 17β-estradiol as a model system. Microscale thermophoresis, apta-PCR affinity assay and surface plasmon resonance were explored to evaluate the reported aptamers’ binding properties in terms of affinity and specificity, and were demonstrated to be successfully applied to the analysis of aptamer-small molecule interactions.

In Vivo Dynamic Monitoring of Small Molecules with Implantable Polymer-Dot Transducer.

Small molecules participate extensively in various life processes. However, specific and sensitive detection of small molecules in a living system is highly challenging. Here, we describe in vivo real-time dynamic monitoring of small molecules by a luminescent polymer-dot oxygen transducer. The optical transducer combined with an oxygen-consuming enzyme can sensitively detect small-molecule substrates as the enzyme-catalyzed reaction depletes its internal oxygen reservoir in the presence of small molecules. We exemplify this detection strategy by using glucose-oxidase-functionalized polymer dots, yielding high selectivity, large dynamic range, and reversible glucose detection in cell and tissue environments. The transducer-enzyme assembly after subcutaneous implantation provides a strong luminescence signal that is transdermally detectable and continuously responsive to blood glucose fluctuations for up to 30 days. In view of a large library of oxygen-consuming enzymes, this strategy is promising for in vivo detection and quantitative determination of a variety of small molecules.

Highly Sensitive Detection of Small Molecule Markers By Surface Plasmon Resonance Imaging

Surface plasmon resonance (SPR) spectroscopy is a label-free method for monitoring binding events among biomolecules in real time and for sensing applications.1 SPR spectroscopy has been widely used in chemistry, biochemistry, and biology research. Our objective is to achieve ultra-low level detection of small molecules that are important markers of cancer disease. Also, we are interested in monitoring pH changes in less abundant biological samples. As we know, pH is an essential parameter in biological, medical and industrial applications. Monitoring the pH level in biological fluids reflects the abnormal conditions in human body.2 , 3We will present our findings on sensitive detection of small molecules and amplification of SPR signals by using chemical indicators and detection by an SPR-array imager Acknowledgements.This project was supported by Oklahoma State University. 1. Fasoli, J. B.; Corn, R. M., Surface Enzyme Chemistries for Ultrasensitive Microarray Biosensing with SPR Imaging. Langmuir 2015, 10.1021/la504797z. 2. Sim, J.; Kwon, D.-S.; Kim, J., Acid-sensitive pH sensor using electrolysis and a microfluidic channel for read-out amplification. RSC Advances 2014, 4 (75), 39634-39638. 3. Kick, A.; Mertig, M., Characterization of pH-sensitive polymer layers by surface plasmon resonance and quartz crystal microbalance. physica status solidi (a), 2015, 10.1002/pssa.201431604.

Enzymatic cleavage and mass amplification strategy for small molecule detection using aptamer-based fluorescence polarization biosensor

Fluorescence polarization (FP) assays incorporated with fluorophore-labeled aptamers have attracted great interest in recent years. However, detecting small molecules through the use of FP assays still remains a challenge because small-molecule binding only results in negligible changes in the molecular weight of the fluorophore-labeled aptamer. To address this issue, we herein report a fluorescence polarization (FP) aptamer assay that incorporates a novel signal amplification strategy for highly sensitive detection of small molecules. In the absence of adenosine, our model target, free FAM-labeled aptamer can be digested by nuclease, resulting in the release of FAM-labeled nucleotide segments from the dT-biotin/streptavidin complex with weak background signal. However, in the presence of target, the FAM-labeled aptamer–target complex protects the FAM-labeled aptamer from nuclease cleavage, allowing streptavidin to act as a molar mass amplifier. The resulting increase in molecular mass and FP intensity of the aptamer–target complex provides improved sensitivity for concentration measurement. The probe could detect adenosine from 0.5μM to 1000μM, with a detection limit of 500nM, showing that the sensitivity of the probe is superior to aptamer-based FP approaches previously reported for adenosine. Importantly, FP could resist environmental interferences, making it useful for complex biological samples without any tedious sample pretreatments. Our results demonstrate that this dual-amplified, aptamer-based strategy can be used to design fluorescence polarization probes for rapid, sensitive, and selective measurement of small molecules in complicated biological environment.

Nanostructure-initiator mass spectrometry (NIMS) for molecular mapping of animal tissues.

Nanostructure-initiator mass spectrometry (NIMS) is an established method for sensitive detection of small molecules in complex samples. It is based on the optimal combination of a porous Si substrate and a carefully selected polymer coating to allow certain analytes of interest to be concentrated on the substrate for effective ionization with minimal background interference from conventional organic matrices. The previous chapter has detailed the history and current state of the art of the technique in small-molecule profiling and imaging applications. We describe here a simple step-by-step protocol for substrate fabrication and sample preparation that provides a starting point for the technique to be adapted and optimized for 2-D biological imaging applications.

Adsorption of Rhodamine 6G on SiO2 and Ag@SiO2 Porous Solids: Coupling Thermodynamics and Raman Spectroscopy

The sensitive detection of small molecules analytes at very low concentrations is of great interest, notably in the medical and environmental fields. In this study, two porous solids, namely SiO2 (dpore ≈ 5 nm) and Ag@SiO2, were prepared. In the latter, the incorporation of Ag nanoparticles in the porous matrix was highlighted using different techniques. Around 0.25 wt % of nanoparticles were inserted with a mean diameter close to 3 nm. Then, the adsorption of rhodamine 6G (R6G) on these two materials was studied coupling thermodynamics and Raman spectroscopy. From a thermodynamic point of view, it was shown that whereas the adsorbed amounts of R6G (Nads) were not affected by the presence of Ag nanoparticles, the adsorption enthalpies in the case of Ag@SiO2 are doubled compared to those of SiO2, highlighting the strong interaction of R6G with the Ag nanoparticle surface. Furthermore, for Nads > 1 μmol g–1, it was evidenced that the Raman response of R6G, in terms of band positions and intensities, is directly related to the amounts of R6G adsorbed on the solid surface and not to the presence of Ag. This result highlights the preconcentration properties of porous solids. For Nads = 0.05 μmol g–1, the intensities of Raman bands that are detected, corresponding to bending modes of ethylamine groups, are enhanced in the case of Ag@SiO2 by a factor of roughly 10. This value corresponds to the order of magnitude generally assumed for the chemical enhancement mechanism of surface enhanced Raman spectroscopy effect. This result and the high values of integral enthalpies of adsorption determined for Ag@SiO2 highlight strong interactions between amine groups and the Ag surface.

Label-free and sensitive electrochemiluminescence aptasensor for the determination of 17β-estradiol based on a competitive assay with cDNA amplification

A label-free competitive electrochemiluminescence (ECL) aptasensor for the determination of 17β-estradiol (E2) was developed based on the immobilization of thiol-capped E2 aptamers onto a gold electrode. A complementary DNA (cDNA) was designed to be used as a detection probe to bind with any unbound E2 aptamer. Tris(2,2′-bipyridyl)ruthenium(II) (Ru(bpy)32+) was used as an ECL indicator and was electrostatically bound to the E2 aptamer; it competes with the cDNA for aptamer binding sites. With less E2, increased amounts of cDNA can bind to the E2 aptamer and adsorb more Ru(bpy)32+, greatly enhancing the ECL intensity. With more E2, the aptamer will capture E2 and bind less cDNA with an accompanying decrease in Ru(bpy)32+ and a decrease in ECL intensity. Thus, a competitive aptasensor based on the specific binding of the E2 aptamer to E2 and cDNA was achieved. The biosensor for E2 possesses a widely linear detection range from 0.01 to 10 nmol L−1 with a detection limit of 1.1 × 10−12 mol L−1 (S/N = 3). The application of the present protocol was demonstrated by determining E2 in human serum, human urine, and tap water samples with recoveries of 89.8–100.0%, 90.0–103.5% and 89.5–95.0%, respectively. The proposed method provides a powerful tool for the rapid and sensitive detection of small molecules in biological and environmental samples.

Enhanced collective optical response of vast numbers of silver nanoparticles assembled on a microbead

We investigated the optical response of a huge number of silver nanoparticles (AgNPs) densely assembled on an organic microsphere, i.e., AgNP-fixed bead, under the collective phenomena of localized surface plasmons. For this purpose, various optical properties of such a AgNP-fixed bead were analyzed in aqueous solution by dark-field optical microscopy and laser Raman microscopy. In particular, in comparison with the optical spectrum of single AgNPs, significant spectral broadening and redshift were observed due to plasmonic superradiance with decreasing interparticle distance to the subnanoscale when using small binder molecules in the AgNP-fixed bead. Furthermore, we observed surface-enhanced Raman scattering and clarified the sensitivity of the signal intensity to the size of the binder molecules between the AgNPs, which can be explained based on optical response theory using a discrete integral with spherical cells. These results and discussion provide a guiding principle for broadband plasmonic light absorbers and for highly sensitive detection of small molecules and nanoscale biomaterials based on vast numbers of nanogaps produced by a bottom-up self-assembly process.

Hapten-Grafted Graphene as a Transducer for Homogeneous Competitive Immunoassay of Small Molecules

A hapten-grafted graphene-based biosensor by integrating both the graphene nanosheets and immunoassay sensing technologies was developed for ultrasensitive homogeneous competitive immunoassay of small molecules. The structure of hapten-grafted graphene avoids the activity loss of biomolecules immobilized onto the graphene surface and is beneficial to preserve the binding affinity between small molecule and its specific antibody. The sandwich structure formed between hapten-grafted graphene nanosheets and fluorescence-labeled antibody increases the quenching efficiency of the organic dye, thereby resulting in high signal-to-background ratios and improved sensitivity for Bisphenol A (BPA) detection. On the basis of fluorescence resonance energy transfer (FRET) and homogeneous competitive immunoassay mechanism, high BPA concentrations in the sample reduce the amount of fluorescence-labeled anti-BPA antibody bound to graphene-BPA nanosheets, thus resulting in remarkable fluorescence signals. The linear quantification of BPA over concentration ranges from 0.5 to 50 nM with a detection limit determined as 0.12 nM. These findings show that the proposed method provides a powerful tool for the rapid and sensitive detection of small molecules in biological and environmental samples.

Target-Triggered Cyclic Assembly of DNA–Protein Hybrid Nanowires for Dual-Amplified Fluorescence Anisotropy Assay of Small Molecules

Aptamer-based fluorescence anisotropy (FA) assays have attracted great interest in recent years. However, a key factor that determines FA value is molar mass, thus limiting the utility of this assay for the detection of small molecules. To solve this problem, streptavidin, as a molar mass amplifier, was used in a hybridization chain reaction (HCR) to construct a target-triggered cyclic assembly of DNA-protein hybrid nanowires for highly sensitive detection of small molecules by fluorescence anisotropy. In this assay, one blocking DNA strand is released by target-aptamer recognition. The DNA then serves as an initiator to trigger enzyme-free autonomous cross-opening of hairpin probes via HCR to form a DNA nanowire for further assembly of streptavidin. Using adenosine triphosphate (ATP) as the small molecule target, this novel dual-amplified, aptamer-based FA assay affords high sensitivity with a detection limit of 100 nM. This limit of detection (LOD) is much lower than that of the disassembly approach without HCR amplification or the assembly strategy without streptavidin. In contrast to the previous turn-off disassembly approaches based on nonspecific interactions between the aptamer probe and amplification moieties, the proposed aptamer-based FA assay method exhibits a turn-on response to ATP, which can increase sensing reliability and reduce the risk of false hits. Moreover, because of its resistance to environmental interferences, this FA assay has been successfully applied for direct detection of 0.5 μM ATP in complex biological samples, including cell media, human urine, and human serum, demonstrating its practicality in real complex biological systems.

Signal-amplification detection of small molecules by use of Mg2+- dependent DNAzyme

Because small molecules can be beneficial or toxic in biology and the environment, specific and sensitive detection of small molecules is one of the most important objectives of the scientific community. In this study, new signal amplification assays for detection of small molecules based on Mg(2+)-dependent DNAzyme were developed. A cleavable DNA substrate containing a ribonucleotide, the ends of which were labeled with black hole quencher (BHQ) and 6-carboxyfluorescein (FAM), was used for fluorescence detection. When the small molecule of interest is added to the assay solution, the Mg(2+)-dependent DNAzyme is activated, facilitating hybridization between the Mg(2+)-dependent DNAzyme and the DNA substrate. Binding of the substrate to the DNAzyme structure results in hydrolytic cleavage of the substrate in the presence of Mg(2+) ions. The fluorescence signal was amplified by continuous cleavage of the enzyme substrate. Ochratoxin A (OTA) and adenosine triphosphate (ATP) were used as model analytes in these experiments. This method can detect OTA specifically with a detection limit as low as 140 pmol L(-1) and detect ATP specifically with a detection limit as low as 13 nmol L(-1). Moreover, this method is potentially extendable to detection of other small molecules which are able to dissociate the aptamer from the DNAzyme, leading to activation of the DNAzyme.

Sensitive Detection of Small Molecules by Competitive Immunomagnetic-Proximity Ligation Assay

A novel detection method of small molecules, competitive immunomagnetic-proximity ligation assay (CIPLA), was developed and described in this report. Through the proximity effect caused by special proximity probes we prepared, small molecules can be detected using only one monoclonal antibody. CIPLA overcomes the obstacle that the proximity ligation assay (PLA) cannot be used in small molecular detection, as two antibodies are unable to combine to one small molecule due to its small molecular structure. Two small molecular compounds, clenbuterol (CLE) and ractopamine (RAC), were selected as targets for CIPLA. The limit of detection (LOD) reached 0.01 ng mL(-1), which was 10-50-fold lower than ELISA. With 5 orders of magnitude of the dynamic range achieved, the excellent sensitivity and broad dynamic range of CIPLA are noted. It can be applied widely in the sensitive detection of many other small molecular materials such as pesticides, additives in food, drugs, and biological samples, which have great significance in both theoretical and practical aspects.

High-Salt-Tolerance Matrix for Facile Detection of Glucose in Rat Brain Microdialysates by MALDI Mass Spectrometry

Due to its strong ultraviolet absorption, high salt tolerance, and little interference in the low molecular weight region, N-(1-naphthyl) ethylenediamine dihydrochloride (NEDC) has been applied as a matrix to measure the level of glucose in rat brain microdialysates by matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) in combination with in vivo microdialysis. By monitoring the ion signals of (glucose + Cl)(-) in the mass spectra, we achieved a low detection limit of ~10 μM for glucose in 126 mM NaCl, which is a typical component in artificial cerebrospinal fluid, without prior sample purification. It is concluded that NEDC-assisted laser desorption/ionization (LDI) MS is a fast and general method for sensitive detection of small molecules (such as glucose and amino acids) in high ionic strength solutions.

Time-resolved fluorescence biosensor for adenosine detection based on home-made europium complexes

In this protocol, the authors report a time-resolved fluorescence biosensor based on home-made europium complexes for highly sensitive detection of small molecules using adenosine as a model analyte. The fluorophore that used is europium complexes. Its signal can be measured in a time-resolved manner that eliminates most of the unspecific fluorescent background. The amino modified aptamer probe, which is designed to specifically recognize adenosine, is combined to the aldehyde-group modified glass slide by covalent bond. Europium complex-labeled a short ssDNA, designed to segment hybridize with aptamer probe is immobilized on the glass slide by hybridization reaction. In the presence of adenosine, the aptamer part is more inclined to bounds with adenosine and triggers structure-switching of the aptamer from aptamer/ssDNA duplex to aptamer/target complex. As a result, europium complexes-labeled ssDNA is forced to dissociate from the sensor interface, resulting in time-resolved fluorescence intensity decrease. The decrement intensity is proportional to the amount of adenosine. Under optimized assay conditions, a linear range (1.0×10−8M to 1.0×10−7M) is got with low detection limit of 5.61nM. The biosensor exhibits excellent selectivity and can provide a promising potential for aptamer-based adenosine detection.

Direct surface plasmon resonance immunosensor for in situ detection of benzoylecgonine, the major cocaine metabolite

In this paper the development of the first direct surface plasmon resonance (SPR) immunoassay for the detection of benzoylecgonine (BZE) is described. Immunosensor chips consisting of a high affinity monoclonal anti-BZE-antibody (anti-BZE–Ab) immobilized at high density to a sensor chip were prepared. First, BZE detection in Hepes buffer was achieved by direct, real time monitoring of the binding between BZE in solution and the surface bound antibody. The detection protocol was based on calibration curves obtained from reaction rate data and end point data analysis of sensorgrams registered after injection of a series of known BZE concentrations over the chips. Moreover, immunosensor accuracy, reproducibility, stability and robustness were tested to demonstrate their good performance as reusable devices. The immunosensor was used for BZE detection in oral fluid (OF) showing that, within 180 s, our immunoassay detects BZE concentrations as low as 4 μg/L in filtered OF-buffer (1:4) samples. This value is remarkably lower than current cut off levels established by the Substance Abuse and Mental Health Services Administration. These results manifest the potential use of this direct SPR immunoassay for the in situ sensitive detection of recent cocaine abuse, of utility in roadside drug OF testing. Moreover, it exemplifies the high potential of direct SPR immunoassays for the rapid, sensitive detection of small molecules in contrast with the more established indirect methods.

Noncompetitive phage anti-immunocomplex real-time polymerase chain reaction for sensitive detection of small molecules.

Immuno polymerase chain reaction (IPCR) is an analytical technology based on the excellent affinity and specificity of antibodies combined with the powerful signal amplification of polymerase chain reaction (PCR), providing superior sensitivity to classical immunoassays. Here we present a novel type of IPCR termed phage anti-immunocomplex assay real-time PCR (PHAIA-PCR) for the detection of small molecules. Our method utilizes a phage anti-immunocomplex assay (PHAIA) technology in which a short peptide loop displayed on the surface of the M13 bacteriophage binds specifically to the antibody-analyte complex, allowing the noncompetitive detection of small analytes. The phagemid DNA encoding this peptide can be amplified by PCR, and thus, this method eliminates hapten functionalization or bioconjugation of a DNA template while providing improved sensitivity. As a proof of concept, two PHAIA-PCRs were developed for the detection of 3-phenoxybenzoic acid, a major urinary metabolite of some pyrethroid insecticides, and molinate, a herbicide implicated in fish kills. Our results demonstrate that phage DNA can be a versatile material for IPCR development, enabling universal amplification when the common element of the phagemid is targeted or specific amplification when the real time PCR probe is designed to anneal the DNA encoding the peptide. The PHAIA-PCRs proved to be 10-fold more sensitive than conventional PHAIA and significantly faster using magnetic beads for rapid separation of reactants. The assay was validated with both agricultural drain water and human urine samples, showing its robustness for rapid monitoring of human exposure or environmental contamination.

Blank peak current-suppressed electrochemical aptameric sensing platform for highly sensitive signal-on detection of small molecule

In this contribution, an electrochemical aptameric sensing scheme for the sensitive detection of small molecules is proposed using adenosine as a target model. A ferrocene (Fc)-functionalized thiolated aptamer probe is adapted and immobilized onto an electrode surface. Introducing a recognition site for EcoRI into the aptamer sequence not only suppresses the peak current corresponding to blank sample but also provides a signal-on response mechanism. In the absence of adenosine, the aptamer can fold into a hairpin structure and form a cleavable double-stranded region. Fc is capable of being removed from electrode surface by treatment with endonuclease, and almost no peak current is observed. The adenosine/aptamer binding induces the conformational transition of designed aptamer, dissociating the cleavable double-stranded segment. Therefore, the integrated aptamer sequence is maintained when exposing to endonuclease, generating a peak current of Fc. Utilizing the present sensing scheme, adenosine even at a low concentration can give a detectable current signal. Thus, a detection limit of 10−10 M and a linear response range from 3.74 × 10−9 to 3.74 × 10−5 M are achieved. The proposed proof-of-principle of a novel electrochemical sensing is expected to extend to establish various aptameric platforms for the analysis of a broad range of target molecules of interest.