Label-free Fluorescent Aptasensor Research Articles

Label-free fluorescent aptasensor for myoglobin detection based on an aggregation-induced emission enhancement imidazole compound and a Fe3O4@4-VP platform

Acute myocardial infarction (AMI) is recognized as one of the deadliest cardiovascular diseases in the world, causing serious public health problems worldwide. Myoglobin (Mb) is an important biomarker for AMI. Therefore, the accurate detection of Mb is crucial for the early detection and clinical diagnosis of AMI. In this study, a novel imidazole Compound C and novel Fe3O4@4-VP microspheres were prepared and used to construct an unlabeled fluorescence aptasensor for Mb detection. Compound C can bind to the aptamer of Mb (Mb-Apt) through electrostatic attraction to form a C/Mb-Apt complex and produce the aggregation-induced emission enhancement effect. Then, Mb was added to the C/Mb-Apt solution. Mb could combine with C/Mb-Apt to form a C/Mb-Apt/Mb complex. Subsequently, Fe3O4@4-VP was added to the above mixture. The bases of Mb-Apt already combined with Mb, and thus, C/Mb-Apt/Mb would no longer interact with the pyridine rings on the surface of Fe3O4@4-VP through π-π stacking. After magnetic separation, the C/Mb-Apt/Mb complex remained in the supernate. The supernate exhibited strong fluorescence and its fluorescent intensity was proportional to the level of Mb. Experimental results showed an excellent linear relationship between the fluorescent intensity of the supernate and the level of Mb within the range of 0.167–20 ng/mL, with a detection limit reaching as low as 0.05 ng/mL (S/N = 3). In addition, the aptasensor exhibited high specificity and could be used for detecting Mb in human serum.

Label-free fluorescence aptasensor for the detection of cadmium(II) ion based on the conformational switching of aptamer and thioflavine T.

A simple label-free Cd2+ fluorescent aptasensor was proposed using aptamer as a recognition element and thioflavine T (ThT) as a signal reporter. The presence of Cd(II) can induce the conformational switching of the aptamer probe, accompanied by a change in fluorescence intensity. According to the difference in fluorescence signals before and after the addition of Cd2+, a fluorescence sensor for Cd2+ assay was established. Under the better experimental conditions, the sensor displayed a good linear range from 2 to 50nM and the excellent detection limit was 0.8nM. The method demonstrated high sensitivity and good selectivity. The aptasensor could detect Cd2+ in simulated water samples with satisfactory results. The proposed method had obvious advantages that was without chemical modification of fluorescent groups and complicated target preconcentration. It provided a new analytical platform for the detection of heavy metal ion pollution in environmental and biomedical fields.

Label-free and low-background fluorescent structure-switching aptasensor for sensitive detection of staphylococcal enterotoxin A based on graphene oxide-assisted separation of ssDNA

Label-free and low-background fluorescent structure-switching aptasensor for sensitive detection of staphylococcal enterotoxin A based on graphene oxide-assisted separation of ssDNA

A label-free ratiometric fluorescent aptasensor based on a peroxidase-mimetic multifunctional ZrFe-MOF for the determination of tetrodotoxin.

A Fe/Zr bimetal-organic framework (ZrFe-MOF) is utilized to establish a ratiometric fluorescent aptasensor for the determination of tetrodotoxin (TTX). The multifunctional ZrFe-MOF possesses inherent fluorescence at 445nm wavelength, peroxidase-mimetic activity, and specific recognition and adsorption capabilities for aptamers, owing to its organic ligand, and Fe and Zr nodes. The peroxidation of o-phenylenediamine (OPD) substrate generates fluorescent 2,3-diaminophenazine (OPDox) at 555nm wavelength, thus quenching the inherent fluorescence of ZrFe-MOF because of the fluorescence resonance energy transfer (FRET) effect. TTX aptamers, which are absorbed on the material surface without immobilization or fluorescent labeling, inhibit the peroxidase-mimetic activity of ZrFe-MOF. It causes the decreased OPDox fluorescence at 555nm wavelength and the inverse restoration of ZrFe-MOF fluorescence at 445nm wavelength. With TTX, the aptamers specifically bind to TTX, triggering rigid complex release from ZrFe-MOF surface and reactivating its peroxidase-mimetic activity. Consequently, the two fluorescence signals exhibit opposite changes. Employing this ratiometric strategy, the determination of TTX is achieved with a detection limit of 0.027ng/mL and a linear range of 0.05-500ng/mL. This aptasensor also successfully determines TTX concentrations in puffer fish and clam samples, demonstrating its promising application for monitoring trace TTX in food safety.

Evolution of high-affinity aptamer probe for label-free and probe conformation switch-based fluorescent biosensing of β-carotene in seaweeds

β-Carotene is an important bioactive component showing great activities and receiving great attention in recent years. The traditional methods for the accurate detection of β-carotene in food are time-consuming. There is an urgent need to establish rapid quantification methods for β-Carotene. Herein, a label-free fluorescent aptasensor which was based on the conformation-switch of the aptamer probe was developed to detect β-carotene in seaweeds. Firstly, a post-SELEX strategy based on extraction and terminal-fixation was established to evolve the anti-β-carotene aptamer to achieve high affinity. An aptamer probe (β-2-TF) with a 177.5-fold affinity increment was obtained and then applied to develop a probe conformation-switch-based fluorescent aptasensor with the assistance of SYBR Green I (SGI). Under the optimal conditions, the aptasensor exhibited good accuracy and selectivity, with a linear detection range of 62.5–4000 nM and a limit of detection (LOD) as low as 39.64 nM. The developed strategies can act as robust references for the analytical method development in the food field.

Label-free competitive time-resolved fluorescent aptasensor for the detection of Pb2+ using ssDNA-sensitized fluorescence of Tb3+ ions

Lead (Pb2+) is one of the most toxic heavy metals, and its environmental pollution and serious damages is a global concern. Therefore, it is necessary to develop effective sensing methods. This study describes a new strategy for the design of label-free competitive time-resolved fluorescent (TRF) aptasensor for detecting Pb2+ ions. The sensing principle of this aptasensor is the competition between Pb2+ and Tb3+ ions to bind to the guanine/thymine-rich sequence (lead aptamer) and the sensitized luminescence of Tb3+ by this sequence. The developed TRF aptasensor demonstrated a good linear detection range from 2.5 nM to 150 nM and a limit of detection (LOD) of 645 pM. In addition, the proposed TRF aptasensor has a high selectivity towards Pb2+, and it has also been successfully utilized to detect this ion in milk and human serum samples. This TRF aptasensor offers advantages such as short analysis time, simple operation, low cost, being label-free, and surpassing the interference of background fluorescence of biological samples due to its TRF characteristic. In this paper, we propose a Tb3+/guanine/thymine-rich sequence system for designing different aptasensors against diverse targets by applying a proper guanine-rich sequence.

Fluorescent polythymidine-templated copper nanoclusters aptasensor for sensitive detection of tropomyosin in processed shrimp products

Tropomyosin (TM) is the main allergen in shellfish. Developing a novel, simple and accurate method to track and detect TM in food products is necessary. In this work, a label-free fluorescent aptasensor based on polythymidine (poly(T))-templated copper nanoclusters (CuNCs) was designed for sensitive detection of TM in processed shrimp products. Magnetic beads (MBs), aptamer and cDNA were used to construct an MBs-aptamer@cDNA complex as a detection probe, and with the presence of TM, the poly(T)-templated CuNCs attached at the end of the cDNA as the fluorescent signal was released from the complex to turn on the fluorescence. Under optimal conditions, the poly(T)-templated CuNCs aptasensor achieved a linear range from 0.1 to 50 μg/mL (R2 = 0.9980), a low limit of detection of 0.0489 μg/mL and an excellent recovery percentage of 105.29%-108.91% in the complex food matrix, providing a new approach for food safety assurance.

Fluorometric detection of alpha-fetoprotein based on the use of a novel organic compound with AIE activity and aptamer-modified magnetic microparticles

BackgroundLiver cancer is one of the most common cancers in the world, and it seriously threatens human life and health. Alpha-fetoprotein (AFP), as a carcinogenic glycoprotein, is an important serum marker for detecting liver cancer. Therefore, the accurate and sensitive determination of AFP is crucial for the early diagnosis and treatment of liver cancer. To this end, a label-free fluorescence aptasensor for detecting AFP based on the use of a novel organic Compound D with an aggregation-induced emission activity and aptamer-modified magnetic microparticles was constructed. ResultsCompound D could combine with the complementary short chain of the aptamer (CSC-Apt) of AFP to form the D/CSC-Apt complex and realize the fluorescence enhancement of Compound D. Then, magnetic particles modified by the Apt of AFP (Apt-Fe3O4) were prepared. When AFP (or nontarget substance) and D/CSC-Apt were successively added to the Apt-Fe3O4 solution, Apt-Fe3O4 selectively bound to AFP or the D/CSC-Apt complex. Magnetic separation technology showed the changes in the fluorescence intensity of the supernatant. The research results revealed a good linear relationship between the changes in the fluorescence intensity of the supernatant and concentration of AFP within the concentration range of 10–10000 pg mL−1. The proposed aptasensor could achieve high-sensitivity and high-specificity detection of AFP, and its limit of detection was 3 pg mL−1 (S/N = 3). Significance and noveltyThe sensor combines the advantages of high selectivity of Apt, high sensitivity of fluorescence analysis, AIE effect and good water solubility of Compound D, and rapid separation using magnetic separation technology. And it can be directly used for the detection of AFP in actual serum samples with high accuracy, whereas most of the methods reported in the literature can only detect AFP in spiked serum samples.

A label-free carbon dots-based fluorescent aptasensor for the detection of V. cholerae O139

The V. cholerae O139 contamination in the environmental water sources causes a greater threat to humans. In this study, a sensitive and label-free aptamer-based fluorescent biosensor was successfully developed. The aptamer specifically identifies the V. cholerae O139 and the quantum dots (QD) used as the fluorescent probe. The aptamer adsorbed to the QD through electrostatic interactions. The quantum dots emitted blue fluorescent at 430 nm with a quantum yield of 45.3 %, which could be effectively quenched on the binding upon the target pathogen. The sensor's fluorescence signal has a linear relationship with the concentration of V. cholerae O139 with a 426 CFU/mL detection limit. Besides, the aptasensor has been tested in detecting V. cholerae O139 through recovery studies using spiked water samples. The recovery of water samples ranges from 85.2 % and 93.46 %, and the relative standard deviation was from 1.13 % to 3.86 %. The result indicated the reliability and reproducibility of the aptasensor in the on-field detection of the pathogen.

An enzyme-free and label-free fluorescent aptasensor for sensitive detection of kanamycin in milk samples based on hybridization chain reaction

An enzyme-free and label-free fluorescent aptasensor for sensitive detection of kanamycin in milk samples based on hybridization chain reaction

Molecular Docking Insight into the Label-Free Fluorescence Aptasensor for Ochratoxin A Detection.

Ochratoxin A (OTA) is the most common mycotoxin and can be found in wheat, corn and other grain products. As OTA pollution in these grain products is gaining prominence as a global issue, the demand to develop OTA detection technology has attracted increasing attention. Recently, a variety of label-free fluorescence biosensors based on aptamer have been established. However, the binding mechanisms of some aptasensors are still unclear. Herein, a label-free fluorescent aptasensor employing Thioflavin T (ThT) as donor for OTA detection was constructed based on the G-quadruplex aptamer of the OTA aptamer itself. The key binding region of aptamer was revealed by using molecular docking technology. In the absence of the OTA target, ThT fluorescent dye binds with the OTA aptamer to form an aptamer/ThT complex, and results in the fluorescence intensity being obviously enhanced. In the presence of OTA, the OTA aptamer binds to OTA because of its high affinity and specificity to form an aptamer/OTA complex, and the ThT fluorescent dye is released from the OTA aptamer into the solution. Therefore, the fluorescence intensity is significantly decreased. Molecular docking results revealed that OTA is binding to the pocket-like structure and surrounded by the A29-T3 base pair and C4, T30, G6 and G7 of the aptamer. Meanwhile, this aptasensor shows good selectivity, sensitivity and an excellent recovery rate of the wheat flour spiked experiment.

Robust tag-free aptasensor for monitoring of tobramycin: Architecting of rolling circle amplification and fluorescence synergism

With the unpredictable risks on human health and ecological safety, tobramycin (TOB) as an extensively applied antibiotic has embraced global concern. Herein, a label-free fluorescent aptasensor was developed that opened up an innovative sensing strategy for monitoring trace TOB levels. Based on the rolling circle amplification (RCA) process, a giant DNA building was established by the catalytic action of T4 DNA ligase and Phi 29 DNA polymerase with the cooperation of the specific aptamer as a primer skeleton. By having the role of signal amplifier template, the RCA product with the G-quadruplex sequence duplications was decorated by a high number of the thioflavin T (ThT) fluorescent dyes. The aptasensor with good selectivity toward TOB achieved a detection limit as low as 150 pM. Thanks to its accurate target quantification, ease of operation, economic manufacture, as well as high potency for real-time and point-of-care testing, the represented aptasensor is superb for clinical application and food safety control.

Screening and identification of a DNA aptamer to construct the label-free fluorescent aptasensor for ultrasensitive and selective detection of clothianidin residue in agricultural products

Clothianidin pesticide not only pollutes the ecological environment, but also poses a potential threat to human health. Thus, it is of great importance to develop efficient and accurate methods to recognize and detect clothianidin residues in agricultural products. Aptamer has the advantages of easy modification, high affinity and good stability, which is particularly suitable as a recognition biomolecule for pesticide detection. However, the aptamer against clothianidin has not been reported. Herein, the aptamer (named CLO-1) had good selectivity and strong affinity (Kd = 40.66 ± 3.47 nM) to clothianidin pesticide, which was screened for the first time by Capture-SELEX strategy. The binding effect of CLO-1 aptamer to clothianidin was further studied by circular dichroism (CD) spectroscopy and molecular docking technique. Finally, the CLO-1 aptamer was used as the recognition molecule to construct a label-free fluorescent aptasensor using GeneGreen dye as sensing signal for the highly sensitive detection of clothianidin pesticide. The constructed fluorescent aptasensor had the limit of detection (LOD) as low as 5.527 μg L−1 for clothianidin, and displayed good selectivity against other competitive pesticides. The aptasensor was applied to detect the clothianidin spiked in tomatoes, pears and cabbages, and the recovery rate was good in the range of 81.99%–106.64%. This study provides a good application prospect for the recognition and detection of clothianidin.

Split aptamer-based fluorescent biosensor for ultrasensitive detection of cocaine using N-methyl mesoporphyrin IX as fluorophore

Cocaine is an extensively used illicit drug worldwide. So, the development of a sensing method for cocaine detection is necessary for examination and lawful action against drug trafficking and abuse. A label-free and simple split aptamer-based fluorescent aptasensor was proposed for cocaine detection using the N-methyl mesoporphyrin IX (NMM)/G-quadruplex system as a fluorescent indicator. The working principle of the developed aptasensor is an increase in the emission intensity of fluorescence of the NMM in the attendance of cocaine due to the formation of split G-quadruplex structure as a suitable platform for attachment of NMM. Under optimized conditions, the designed fluorescent aptasensor exhibited a useful linear concentration range from 50 pM to 25 nM and a limit of detection (LOD) of 16 pM. The applied potential of the designed fluorescent aptasensor was demonstrated by cocaine detection in spiked human serum samples with satisfying recoveries.

A novel label-free dual-mode aptasensor based on the mutual regulation of silver nanoclusters and MoSe2 nanosheets for reliable detection of ampicillin

Current methods for the rapid detection of trace antibiotics in the environment remains problems of low accuracy and false negative or false positive, making the development of fast, and accurate, and reliable methods for antibiotic testing a major challenge that needs to be addressed. Herein, we developed a novel label-free colorimetric and fluorescent dual-mode aptasensor assembled by the strong interaction of layered MoSe2 nanosheets (MoSe2 NSs) with ampicillin (AMP) aptamer functionalized silver nanoclusters (Apt-AgNCs) that specifically bind AMP to allow the sensitive and selective detection of AMP. Apt-AgNCs could be adsorbed on the surface of MoSe2 NSs via van der Waals force to form a nanocomposite, Apt-AgNCs/MoSe2 NSs. Interestingly, Apt-AgNCs/MoSe2 NSs act together to construct dual mode aptasensor through modulation of the intrinsic peroxidase activity of MoSe2 NSs and the fluorescence of Apt-AgNCs. In the presence of AMP, Apt-AgNCs could specifically bind AMP, triggering desorption from the MoSe2 NSs surface, leading to a decrease in the peroxidase activity of the system with the recovery in Apt-AgNCs fluorescence. The dual-signal aptasensor exhibited good linear colorimetric and fluorescence responses in the AMP concentration ranges of 0.115–2.00 μM and 6–100 nM, respectively. Furthermore, the aptasensor was successfully measured AMP levels in commercially-bought milk and lake water with satisfactory results. Unlike single-signal aptasensors, the constructed dual-signal aptasensor could not only improve the detection precision, but also reduce the false positive or false negative results. These promising results suggest that the dual-readout strategy as demonstrated is general mode for the detection of other antibiotics or compounds using various aptamers functionalized AgNCs in concert with MoSe2 NSs.

Docking-aided rational tailoring of a fluorescence- and affinity-enhancing aptamer for a label-free ratiometric malachite green point-of-care aptasensor

Although nucleic acid aptasensors are increasingly applied in the detection of environmentally hazardous biomolecules, several formidable challenges remain with this technique because of their vulnerability, high cost and suboptimal sensitivity. Here, a docking-aided rational tailoring (DART) strategy was established at three levels and in two dimensions for the refinement of malachite green (MG) DNA aptamers. Guided by in silico molecular docking, coarse and fine tailoring were conducted at three levels each, to significantly enhance fluorescence activation intensity and binding affinity in two dimensions. Empowered by the results of the rational tailoring, a mechanistic view of the MG DNA aptamer-target interaction was thoroughly analyzed via four types of interactions. To meet the demand for point-of-care testing (POCT), a label-free and ratiometric fluorescent aptasensor was developed leveraging the tailored MG aptamer, based on the binding site competition-equilibrium effect via the introduction of a reference dye. This sensitive, specific, low-cost and rapid aptasensor subsequently demonstrated outstanding detection performance, achieving an ideal signal response range of 5 nmol·L−1 - 6 μmol·L−1 and a low limit of detection (LOD) of 1.49 nmol·L−1. The DART strategy and systematic exploration of the MG DNA luminescent aptamers herein will provide a valuable reference in the field of aptamer tailoring, biosensing and bioimaging. The proposed label-free ratiometric aptasensor also provides a highly generalizable strategy for hazardous biomolecular detection.

A label-free fluorescent aptasensor based on a novel exponential rolling circle amplification for highly sensitive ochratoxin A detection

Rapid and sensitive analysis of ochratoxin A (OTA) plays an important role in food safety. Here, an aptasensor based on novel exponential rolling circle amplification (ERCA) was proposed for ultrasensitive and label-free fluorescence detection of OTA. The attachment of OTA to its aptamer could release H and rapidly hybridize with CT to initiate rolling circle amplification (RCA). The amplicons could further displace H from APH to initiate recycled RCA, achieving exponential growth of amplification products that contained G4 dimers for lighting up ThT. Benefiting from the exponential amplification efficiency of the ERCA strategy and the high fluorescence quantum yield of G4 dimer/ThT, this strategy exhibited a wide linear range from 10 fg/mL to 10 ng/mL with a detection limit of 4.3 fg/mL. In addition, the aptasensor displayed satisfactory recoveries in real sample analysis. We believe that this novel aptasensor possesses promising application prospects in food safety and medicine detection.

Simultaneous screening of multiple diarrhetic shellfish poisons with group-specific split aptamers and silver nanocluster beacon

Poisoning events concerning diarrhetic shellfish poisons (DSPs) are increasing continually. It is extremely necessary to develop simple analysis methods for screening simultaneously different types of DSPs from food-related samples. Okadaic acid (OA) and its analogues, i.e., dinophysistoxin-1 (DTX-1) and dinophysistoxin-2 (DTX-2), are the prevalent DSPs. Herein, a facile and label-free fluorescent aptasensor targeting the three DSPs was constructed with a pair of group-specific split aptamers and silver nanocluster beacon. In presence of the targets, the DNA templates attached at the ends of the split aptamers would be dragged close to trigger enhanced fluorescence signals from silver nanoclusters. The aptasensor offered high sensitivity and good selectivity, with limit of detection of 2.282 nmolL-1, 19.38 nmolL-1, and 13.61 nmolL-1 for OA, DTX-1, and DTX-2, respectively. Moreover, the applicability of aptasensor was well verified with shellfish and seawater samples. This study provides good reference for further exploration on analysis methods for food-related molecules.

A label-free G-quadruplex aptamer fluorescent aptasensor for visual and real-time kanamycin detection in lake and human samples.

Antibiotic abuse is considered a serious problem affecting human health, necessitating that great attention be paid to explore robust, simple and sensitive methods for rapid evaluation. In this paper, we developed a fluorescent aptasensor for visual and real-time kanamycin detection by taking advantage of the label-free strategy based on H-aggregate disassembly of a chiral cyanine dye induced by a G-quadruplex aptamer. The good sensitivity and selectivity enabled this aptasensor to have a detection limit as low as 43 nM and have high specificity for kanamycin recognition. Furthermore, this assay was successfully applied for the detection of kanamycin in lake water and urine with excellent recoveries.

An inner filter effect-based fluorescent aptasensor for sensitive detection of kanamycin in complex samples using gold nanoparticles and graphene oxide quantum dots.

In this work, a label-free fluorescent aptasensor based on the inner filter effect (IFE) between gold nanoparticles (AuNPs) and graphene oxide quantum dots (GOQDs) was developed for the detection of kanamycin in complex samples. AuNPs are capable of functioning as the fluorescence absorber of GOQDs because of the complementary overlap between their absorption spectra and the emission spectra of GOQDs. AuNPs can effectively quench the fluorescence of GOQDs via the IFE and modulate it with their aggregation state. In the presence of kanamycin, the aptamer is released from the surface of AuNPs, leading to their salt-induced aggregation and the fluorescence recovery of GOQDs. Under the optimum conditions, the fluorescence intensity of GOQDs was linearly proportional to the concentration of kanamycin over the range from 5 to 600 nM, with a detection limit of 3.6 nM. Moreover, the fluorescent aptasensor was successfully applied for kanamycin detection in complex samples (milk, honey and serum), which might hold great promise for kanamycin detection in food safety control and clinical research.