Label-free Fluorescent Aptasensor Research Articles

A simple and label-free fluorescent aptasensor for detection of tobramycin: Appropriate for on-site antibiotic monitoring

Herein a simple fluorescent aptasensor is introduced to quantitatively monitor tobramycin (TOB) antibiotic by using the inherent hairpin structure of TOB aptamer and SYBR Green I (SGI) dye, as the biorecognition element and signal reporter, respectively. In the presence of TOB, the preferential binding of the aptamer towards its target induces the conformational change of the aptamer from hairpin-like to a single string structure. Hence, the SGI intercalation is reduced, decreasing the fluorescent intensity. Under the effect of experimental conditions, there was a wide linear relationship between the relative fluorescence emission and TOB concentration in the range of 0.1–6 µM. Besides, the aptasensor could detect TOB with the detection limit as low as 0.063 µM. The developed aptasensor was utilized to determine TOB in human serum and milk samples with the detection limits of 0.043 and 0.034 µM, respectively. The label-free aptasensor with the main features of feasibility, simplicity and high sensitivity holds the superior potential to develop a low-cost sensing method for human diagnostics, food control, and environmental monitoring. The designed aptasensor can be efficiently applied by non-specialist consumers for on-site monitoring of the diverse targets by substituting their specific aptamers.

A novel fluorescent aptasensor based on exonuclease-assisted triple recycling amplification for sensitive and label-free detection of aflatoxin B1

Aflatoxins are the most toxic type of mycotoxins, which may cause serious carcinogenesis, teratogenesis, and mutagenesis to humans and animals. In this work, we demonstrate a novel label-free fluorescent aptasensor based on exonuclease-assisted triple recycling amplification for the sensitive detection of aflatoxin B1 (AFB1). With the close cooperation of T7 exonuclease and three elaborately designed hairpin probes, the target AFB1 can perform three consecutive cycles of amplification reactions. In this process, each hairpin probe is fully utilized, and the target AFB1, the secondary target and the tertiary target are recycled, thereby achieving a high amplification. Interestingly and importantly, the secondary and tertiary targets generated by amplification are also excellent DNA template sequences for silver nanoclusters (AgNCs). In the presence of NaBH4 and AgNO3, a great number of DNA-AgNCs are synthesized, thereby producing a strong fluorescent signal. Under optimal conditions, the developed aptasensor exhibited high sensitivity to AFB1 with a low detection limit of 0.19 pg mL−1 and a wide dynamic range of 1 × 10−6–1 μg mL−1. In addition, the aptasensor also performed well in the determination of AFB1 in real samples.

Label-free fluorescence aptasensor based on AuNPs and CQDs for the detection of ATP

A label-free fluorescence aptasensor was studied for adenosine triphosphate (ATP) detection that combines low biosensor toxicity with a simplified preparation process. In this study, the aptamer of the gold nanoparticle@aptamer@carbon quantum dot nanostructure could specifically identify ATP, resulting in the change of the fluorescence signal. In order to analyze the performance of the sensor, the effects of the carbon quantum dot (CQD) concentration and centrifugal rate on the stability of the probe were investigated. The results show that the sensor was superior under the 220 µl CQD volume and 2000 rpm centrifugal rate. Furthermore, the linear relationship between the change of the fluorescence signal and ATP concentration is Y = 359.747 + 0.226X within the volume range of 20 µM–280 µM. The correlation coefficient is 0.98, and the detection limit is 20 µM. No obvious fluorescence change was observed in solutions containing other common ions. On the basis of no pollution and simplicity, this sensor demonstrates great potential as a low-cost diagnostic tool for the detection of various targets, particularly for use in the fields of food safety and biomedical diagnostics.

A label-free fluorescent aptasensor based on HCR and G-quadruplex DNAzymes for the detection of prostate-specific antigen.

Prostate specific antigen (PSA) has been considered as the most potential serological biomarker for the early stage detection of prostate cancer. Here, a label-free fluorescence aptasensing strategy for detecting PSA based on hybridization chain reaction (HCR) and G-quadruplex DNAzymes has been developed. This designed strategy consists of three DNA probes, aptamer probe (AP), hairpin probe 1 (H1) and hairpin probe 2 (H2). In the presence of target PSA, the aptamer sequences in AP specifically recognized PSA to form a PSA-aptamer complex, causing an AP conformation change and thus releasing the initiator, which triggered the chain-like assembly of H1 and H2 that yielded extended nicked double-stranded DNA through HCR. Upon the addition of hemin, the G-rich segments at the end of H1 and H2 self-assembled into the peroxidase-mimicking hemin/G-quadruplex DNAzymes, which catalyzed the hydrogen peroxide-mediated oxidation of thiamine to give a fluorescence signal dependent on the concentration of PSA. Under optimal conditions, a limit of detection of 0.05 nM and a linear range from 0.1 nM to 1 nM (R2 = 0.9942) were achieved by this assay. In addition, other interfering proteins, such as IgG, AFP and CEA, did not produce any significant change in the fluorescence intensity response, indicating good selectivity of this sensor for PSA detection. Finally, this proposed aptasensor was successfully used for diluted serum samples.

A Label-free "Lock-key" Fluorescence Aptasensing Based on Triplex-helix DNA and G-quadruplex for CA15-3 Detection.

Herein, we designed a label-free fluorescent aptasensor based on triple-helix DNA and G-quadruplex for carbohydrate antigen (CA15-3) detection. The triplex-helix structure can be formed with inserted G-rich DNA (IG) and aptamer DNA (Apt), which like a "lock" to lock the G-rich sequences. The CA15-3 was the "key", which specifically combined with aptamer sequences of Apt, resulting in liberating IG from the triplex-helix "lock". Then, the G-rich sequences of IG were formed into G-quadruplex and specifically interacted with N-methylmesoporphyrin IX (NMM), which greatly enhanced the fluorescence of the solution. However, when the "key" did not exist, the "lock" was fastened and fluorescence intensity did not change. With this proposed method, the concentration of CA15-3 can be effectively detected from 0.01 to 5 U mL-1 with a detection limit (LOD) of 0.01 U mL-1. Furthermore, this proposed biosensor can be applied to spiked human serum with great precision and reproducibility.

A highly sensitive, simple and label-free fluorescent aptasensor for tobramycin sensing based on PicoGreen intercalation into DNA duplex regions of three-way junction origami

Structure-selective light-up dyes are ideal materials for designing label-free DNA-based biosensors and have gained more interest with researchers. In this study, PicoGreen (PG), as a cyanine fluorogenic dye with a low intrinsic fluorescence emission rate, was employed in a design of an aptasensor based on the formation of three-way junction (3WJ) structure to sense tobramycin (TOB), a member of aminoglycoside antibiotic family with a narrow therapeutic index, in a highly sensitive and simple procedure. In fact, the presence of TOB in the assay changes the number of double-stranded sequences in the engineered design, providing the basis for the difference in the intensity of PG fluorescence signal. The designed biosensor showed a wide linear range from 80 nM to 2 µM with a limit of detection (LOD) equal to 21.86 nM in water. Moreover, it was able to determine TOB in human serum, as a complex matrix, in two linear zones of 5–20 µM and 20–300 µM with a LOD of 321.2 nM, with no need for any complicated sample processing, including addition of organic solvents or ultrafiltering, and only through dilution for 40 times with water. Our results indicated that the prepared sensing strategy could be applicable for clinical samples.

G-quadruplex specific thioflavin T-based label-free fluorescence aptasensor for rapid detection of tetracycline.

A label-free fluorescence aptasensor was developed for the rapid detection of tetracycline (TET) based on G-quadruplex structure of TET aptamers and G-quadruplex specific dye Thioflavin T (ThT). The fluorescence of free ThT is essentially weak in aqueous solution, whereas it selectively identifies the G-quadruplex of aptamers to form the G-quadruplex/ThT conjugates, resulting in an enormous increase of the fluorescence intensity. However, the fluorescence intensity of G-quadruplex/ThT conjugates was drastically suppressed due to the release of free ThT from G-quadruplex/ThT conjugates after the addition of TET via specific binding with TET aptamers. The key factors affecting sensitivity and selectivity including the reaction medium, binding time of ThT to TET aptamers, incubation time between TET aptamers and TET, concentration of ThT and TET aptamers were investigated in detail. The optimal conditions were as follows: ultrapure water as reaction medium, binding time of 5min, incubation time of 1min, 9.0μmol/L ThT and 0.03μmol/L aptamers. A good linear relationship (correlation coefficient of 0.9973) was obtained between the fluorescence quenching efficiency (F0-F)/F0 and the logarithm of TET concentration in the range of 0.01-1.0μmol/L. The limit of detection was 0.001μmol/L (S/N=3). The proposed assay was applied for the detection of TET in the spiked honey and milk samples with recoveries ranging from 93.5% to 106.9%. The developed label-free fluorescence aptasensor showed advantages of high specificity, low cost and short time-consuming, illustrating potential application for on-site detection of TET in foodstuffs.

Exonuclease I-assisted fluorescence aptasensor for tetrodotoxin.

A fluorescence aptasensor for the highly specific and sensitive determination of tetrodotoxin was established with tetrodotoxin-aptamer as the recognition unit, berberine as the signal reporter and exonuclease I as the elimination agent for the background. Berberine has a weak fluorescence emission at 540nm, and it can form the tetrodotoxin-aptamer/berberine complex, resulted in an increased fluorescence. After introducing exonuclease I, it can degrade the single strand oligonucleotides of tetrodotoxin-aptamer into the single nucleotide in the absence of tetrodotoxin, which lead to dramatic fluorescence quenching, and reduce the background signal of sensing system. Once tetrodotoxin is in the presence, tetrodotoxin-aptamer is converted into the stable neck ring conformation, which resists the degradation of exonuclease I and provides a more rigid micro-environment for the excited state of berberine, and then the strong fluorescence is observed. Based on the above properties, an ultrasensitive label-free fluorescence aptasensor for tetrodotoxin is established. The fluorescence aptasensor shows good analytical performance with the linear increase of fluorescence intensity at the tetrodotoxin concentration from 0.030nM to 6.0×103nM. The detection limit of 11.0 pM is much lower than that of other reported sensor methods.

A Label-Free Fluorescent Aptasensor for Detection of Staphylococcal Enterotoxin A Based on Aptamer-Functionalized Silver Nanoclusters.

Staphylococcal enterotoxin A (SEA) is a worldwide public health problem accounting for the majority of food poisoning which is produced by Staphylococcus aureus, threatening human health and leading to various foodborne diseases. Therefore, it is of great significance to develop a sensitive detection method for SEA to ensure food safety and prevent foodborne diseases in humans. In this study, an adaptive fluorescence biosensor for the detection of staphylococcal enterotoxin A (SEA) was designed and developed by combining DNA silver nanoclusters (DNA-AgNCs) with polypyrrole nanoparticles (PPyNPs). Fluorescent AgNCs, synthesized using aptamers as templates, were used as fluorescence probes, whose fluorescence was quenched by PPyNPs. In the presence of the target SEA, DNA-AgNCs were forced to desorb from the surface of PPyNPs through the binding of SEA with the aptamer-DNA-AgNCs, thereby resulting in fluorescence recovery. Under the optimized conditions, the relative fluorescence intensity (FI) showed a linear relationship with the SEA concentration in the range from 0.5 to 1000 ng/mL (Y = 1.4917X + 0.9100, R2 = 0.9948) with a limit of detection (LOD) of 0.3393 ng/mL. The sensor was successfully used to evaluate the content of SEA in milk samples, and the recovery efficiency of SEA was between 87.70% and 94.65%. Thus, the sensor shows great potential for application in food analysis. In short, the proposed platform consisted of an aptamer fluorescent sensor that can be used for the ultrasensitive detection of various toxins by taking advantage of the excellent affinity and specificity of corresponding aptamers.

Detection of aflatoxin B1 with a new label-free fluorescent aptasensor based on exonuclease I and SYBR Gold.

This paper describes a new label-free fluorescent aptasensor for the detection of aflatoxin B1 (AFB1) based upon exonuclease I (Exo I) and SYBR Gold, in which SYBR Gold, aptamer, AFB1, and Exo I were used. Specific combinations of aptamer and AFB1 occurred in the presence of AFB1 and consequently altered the spatial structure of the aptamer, thereby preventing its digestion by Exo I. When SYBR Gold was added, intense fluorescence was observed. Additionally, a good linear relationship was observed under optimized conditions between the fluorescence intensities and the AFB1 concentrations (R2 = 0.993). The established aptamer sensor was highly sensitive and exhibited a low limit of detection of 1.82 ng mL-1, with superior specificity for AFB1. It was also used in the quantification of AFB1 levels in soybean sauce samples and demonstrated satisfactory recoveries in the scope of 94.8-108.9%. The proposed sensor is highly sensitive, low cost, and capable of rapid detection and can thus be used to determine mycotoxin levels in a wide range of feeds and food products in a high-throughput and quantitative means.

A label-free aptasensor for turn-on fluorescent detection of ochratoxin A based on aggregation-induced emission probe

A novel label-free fluorescence aptasensor used for the detection of ochratoxin A (OTA) is presented in this study. When aggregated on the surface of DNA aptamer, aggregation-induced emission (AIE) fluorescence probe presents turn-on fluorescence property. The method proposed in this article was based on an AIE probe, 4, 4-(1E,1E)-2, 2-(anthracene-9, 10-diyl) bis (ethene-2, 1-diyl) bis (N, N, N-trimethylbenzenaminium iodide) (DSAI). With OTA present, the aptamer will combine with OTA and the conformation of the aptamer will switch to an antiparallel G-quadruplex from the initial random coil, which obstructs its digestion by Exo I. After the solution is added with DSAI, DSAI will aggregate on the surface of the aptamer/OTA complex and produces a strong emission. In the range of 5 to 500 ng · ml−1 OTA concentrations, the fluorescence increases with a linear logarithm relationship. The detection limit is 1.9 ng · ml−1. This method was used to detect OTA in spiked real samples, with recoveries and RSDs in the range of 92.2% to 106.3%, and 2.7% to 5.2%, respectively.

A novel label-free fluorescence aptasensor for dopamine detection based on an Exonuclease III- and SYBR Green I- aided amplification strategy

A novel label-free fluorescence aptasensor was established for the detection of dopamine based on dopamine-triggered Exonuclease III- and SYBR Green I-assisted template DNA recycling and a decrease in fluorescence. SYBR Green I was selected as the fluorescent indicator; SYBR Green I fluoresces upon binding to double stranded DNA (dsDNA). The template DNA (tDNA) was designed to contain two regions: one complementary to the complementary strand (cDNA) and the other complementary to the aptamer. Various concentrations of dopamine automatically triggered the Exonuclease III restriction digestion to release SYBR Green I. Consequently, the fluorescence signal significantly decreased. Therefore, a method for detecting the dopamine concentration was established. Polyacrylamide gel electrophoresis was also used to confirm the feasibility of the developed sensor for detecting dopamine. Under optimal conditions, the variation in fluorescence was proportional to the concentration of dopamine in the range of 1.0 × 10-10 M to 10.0 × 10-9 M, with a detection limit of 8.0 × 10-11 M. The developed aptasensor showed good selectivity and was successfully applied to detect dopamine in mouse brain tissues. Due to its remarkable sensitivity and selectivity, the constructed fluorescence aptasensor was applicable not only for the routine detection of dopamine but also for diagnosing disease.

An Enzyme- and Label-Free Fluorescence Aptasensor for Detection of Thrombin Based on Graphene Oxide and G-Quadruplex.

An enzyme- and label-free aptamer-based assay is described for the determination of thrombin. A DNA strand (S) consisting of two parts was designed, where the first (Sa) is the thrombin-binding aptamer and the second (Se) is a G-quadruplex. In the absence of thrombin, Sa is readily adsorbed by graphene oxide (GO), which has a preference for ss-DNA rather than for ds-DNA. Upon the addition of the N-methyl-mesoporphyrin IX (NMM), its fluorescence (with excitation/emission at 399/610 nm) is quenched by GO. In contrast, in the presence of thrombin, the aptamer will bind thrombin, and thus, be separated from GO. As a result, fluorescence will be enhanced. The increase is linear in the 0.37 nM to 50 µM thrombin concentration range, and the detection limit is 0.37 nM. The method is highly selective over other proteins, cost-effective, and simple. In our perception, it represents a universal detection scheme that may be applied to other targets according to the proper choice of the aptamer sequence and formation of a suitable aptamer-target pair.

Determination of dopamine by a label-free fluorescent aptasensor based on AuNPs and carbon quantum dots

In order to reduce the complex modification process and obtain a fast and label-free aptasensor, a label-free fluorescent aptasensor based on gold nanoparticles (AuNPs) and carbon quantum dots (CQDs) was constructed for rapid determination of dopamine (DA). In the absence of DA, CQDs were adsorbed on the surface of AuNPs through the electrostatic force between CQDs and aptamer, which led to the fluorescence quenching of CQDs. When DA were added, they combined with the corresponding aptamers with higher affinity and selectivity, resulting in CQDs far from the surface of AuNPs and thus the fluorescence intensity of CQDs was recovered. Under the optimal condition, the fluorescence intensity of the released CQDs was linearly proportional to the concentration of DA in a range of 5×10-8˜2.5×10-4mol/L with a detection limit of 1×10-8mol/L. Moreover, the aptasensor exhibited a satisfactory detection result of DA in human serum samples. This study provided a more effective method to detect a broad range of targets by using appropriate aptamers, holding great potential in the field of food safety and biomedical diagnostics.

Label-Free Fluorescent Aptasensor for Ochratoxin-A Detection Based on CdTe Quantum Dots and (N-Methyl-4-pyridyl) Porphyrin.

With the widespread contamination of ochratoxin A (OTA), it is of significant importance for detecting OTA in foods and traditional Chinese medicine (TCM). In this study, a novel label-free fluorescent aptasensor utilizing the interaction between OTA-triggered antiparallel G-quadruplex and (N-methyl-4-pyridy) porphyrin (TMPyP) for the rapid and sensitive determination of OTA was established. The fluorescence of CdTe quantum dots (QDs) could be quenched by TMPyP. In the presence of analyte (OTA), the aptamer could recognize OTA and transform from a random coil to the antiparallel G-quadruplex. The interaction between G-quadruplex and TMPyP could release CdTe QDs from TMPyP, and thus recover the fluorescence of CdTe QDs. Under optimized conditions, the detection limit of the designed aptasensor was 0.16 ng mL−1, with a linear range of 0.2 to 20 ng mL−1. Furthermore, this aptasensor showed high selectivity toward OTA against other structural analogs and other mycotoxins, and was successfully applied in Astragalus membranaceus samples. The presented aptasensor for OTA detection could be a promising tool for the field monitoring of food and TCM.

A label-free fluorescent aptasensor for the detection of Aflatoxin B1 in food samples using AIEgens and graphene oxide

The detection of Aflatoxin B1 (AFB1) has attracted extensive attention for food safety is a worldwide public health problem. Herein, a novel, simple and label-free fluorescent aptasensor, based on quaternized tetraphenylethene salt (TPE-Z), graphene oxide (GO) and AFB1 aptamer, has been constructed to detect AFB1. In the presence of AFB1, AFB1 aptamer undergoes a conformational switch from single stranded structure to the AFB1/AFB1 aptamer complex upon target binding, which induces the release of TPE-Z/AFB1 aptamer from the surface of GO. Thus, the fluorescence of TPE-Z/AFB1 aptamer is recovered. The assay can be performed by simply mixing TPE-Z, AFB1 aptamer, the GO and the AFB1 samples with a detection limit of 0.25 ng/mL. It is highly selective against other aflatoxins in foods and its performance has been verified in food samples (corn, milk and rice) with known concentration AFB1.

A label-free and enzyme-free fluorescent aptasensor for sensitive detection of acetamiprid based on AT-rich dsDNA-templated copper nanoparticles

A label-free and enzyme-free aptasensor for sensitive assay of acetamiprid has been established using AT-rich double-stranded (ds) DNA-templated copper nanoparticles (CuNPs) as fluorescent probe. In this work, two hairpin DNA, HP1 and HP2, were elaborately designed with AT-rich DNA sequences in their loops. The aptamer of acetamiprid was located at the 3′-terminal of HP1, which was caged in the stem of HP1. Upon the addition of acetamiprid, the aptamer could combine with acetamiprid to form a target/aptamer complex, and thus its free 5′-terminal was released. Subsequently, the protruded 3′-terminal of HP2 could hybridize with the free 5′-terminal of HP1 to form a stable AT-rich dsDNA. When it interacted with Cu2+ and ascorbic acid (AA), the AT-rich dsDNA/CuNPs were generated with strong fluorescence, offering a “switch-on” detection of acetamiprid. The developed strategy could high selectively detect acetamiprid at the concentration as low as 2.37 nM. Moreover, the possibility of this strategy for the food sample analysis was also investigated. The obtained results demonstrate that the developed strategy has a promising application potential for acetamiprid assay in food safety fields.

A Label-Free Fluorescent AND Logic Gate Aptasensor for Sensitive ATP Detection.

In this study, a label-free fluorescent, enzyme-free, simple, highly sensitive AND logic gate aptasensor was developed for the detection of adenosine triphosphate (ATP). Double-stranded deoxyribonucleic acid (DNA) with cohesive ends was attached to graphene oxide (GO) to form an aptasensor probe. ATP and single-stranded DNA were used as input signals. Fluorescence intensity of PicoGreen dye was used as an output signal. The biosensor-related performances, including the logic gate construction, reaction time, linearity, sensitivity, and specificity, were investigated and the results showed that an AND logic gate was successfully constructed. The ATP detection range was found to be 20 to 400 nM (R2 = 0.9943) with limit of detection (LOD) of 142.6 pM, and the sensitivity range was 1.846 × 106 to 2.988 × 106 M−1. This method for the detection of ATP has the characteristics of being simple, low cost, and highly sensitive.

Label-free fluorescent aptasensor of Cd2+ detection based on the conformational switching of aptamer probe and SYBR green I

A label-free fluorescence aptasensor based on conformational switching of aptamer induced by Cd2+ was established for the good selective determination of Cd2+. The aptamer could act as a recognition element for Cd2+. SYBR green I (SG), as a signal reporter, can bind to the small groove of double-stranded DNA (dsDNA) constituted the aptamer and the complementary strand, establishing the dsDNA-SG complex and generating high fluorescence intensity. In the presence of Cd2+, the specific recognition and binging of aptamers preferentially with Cd2+ induce free of complementary strands from dsDNA and conformational switching of aptamer from dsDNA to a stem-loop structure, fluorescence intensity of system decrease dramatically. Under the optimum experiment parameters, the aptasensor shows a good linear range from 1.12 μg L−1 to 224.82 μg L−1 and the excellent detection limit as low as 0.34 μg L−1. The relative standard deviations (RSD) for determination of Cd2+ (n = 8) were lower than 4%, and the recoveries were in the range of 98.57% ~ 102.49%. The use of fluorescence aptasensor has good selectivity and does not require any sample treatment or target preconcentration for the detection of Cd2+ in real samples.

Label-free aptasensors based on fluorescent screening assays for the detection of Salmonella typhimurium

We report two label-free fluorescent aptasensor methods for the detection of S. typhimurium. In the first method, we have used a ‘‘turn off’’ approach in which the aptamer is first intercalated with SYBR Green I (SG), leading to a greatly enhanced fluorescence signal. The addition of S. typhimurium (approximately 1530–96938 CFU/mL), which specifically binds with its aptamer and releases SG, leads to a linear decrease in fluorescence intensity. The lowest detection limit achieved with this approach was in the range of 733 CFU/mL. In the second method, a ‘‘turn on’’ approach was designed for S. typhimurium through the Förster resonance energy transfer (FRET) between Rhodamine B (RB) and gold nanoparticles (AuNPs). When the aptamer and AuNPs were mixed with RB, the fluorescence of RB was significantly quenched via FRET. The aptamer adsorbs to the AuNP surface to protect them from salt-induced aggregation, which leads to the fluorescence quenching of RB in presence of AuNPs. Upon the addition of S. typhimurium, S. typhimurium specifically binds with its aptamer and loses the capability to stabilize AuNPs. Thus, the salt easily induces the aggregation of AuNPs, resulting in the fluorescence recovery of the quenched RB. S. typhimurium concentrations ranging from 1530 to 96938 CFU/mL with the detection limit of 464 CFU/mL was achieved with this methodology. Given these data, some insights into the molecular interactions between the aptamer and the bacterial target are provided. These aptasensor methods also may be adapted for the detection of a wide variety of targets.