Label-Free and Simple G-quadruplex-based Turn-Off Fluorescence Assay for the Detection of Kanamycin

We have developed a label-free and turn-off fluorescence assay for the determination of kanamycin. The detection system consists of an aptamer for specifically recognizing kanamycin and two auxiliary probes functionalized with two GGG repeats at the 3′ or 5′ ends for signal reporting. Two probes both hybridize with the aptamer and then their G-rich sequences combine to form a G-quadruplex. When thioflavin T, a fluorophore, is bound to the G-quadruplex, the fluorescence intensity of the solution dramatically increases. Upon the addition of the kanamycin, the aptamer–kanamycin binding inhibits the hybridization of two probes and aptamer, and restrains the GGG repeats from getting closer to form the G-quadruplex structure, resulting a significant decrease in the fluorescence intensity. The proposed aptamer-based fluorescent sensing platform showed a linear relationship with the concentration of kanamycin from 0.6 to 20.0?nM. The detection limit was determined to be 0.33?nM. The sensing platform provides resistance to interferences from other antibiotics and can be used to efficiently recognize kanamycin in real samples.     

A sensitive electrochemical aptasensor for ATP detection based on exonuclease III-assisted signal amplification strategy

A target-induced structure-switching electrochemical aptasensor for sensitive detection of ATP was successfully constructed which was based on exonuclease III-catalyzed target recycling for signal amplification. With the existence of ATP, methylene blue (MB) labeled hairpin DNA formed G-quadruplex with ATP, which led to conformational changes of the hairpin DNA and created catalytic cleavage sites for exonuclease III (Exo III). Then the structure-switching DNA hybridized with capture DNA which made MB close to electrode surface. Meanwhile, Exo III selectively digested aptamer from its 3′-end, thus G-quadruplex structure was destroyed and ATP was released for target recycling. The Exo III-assisted target recycling amplified electrochemical signal significantly. Fluorescence experiment was performed to confirm the structure-switching process of the hairpin DNA. In fluorescence experiment, AuNPs–aptamer conjugates were synthesized, AuNPs quenched fluorescence of MB, the target-induced structure-switching made Exo III digested aptamer, which restored fluorescence. Under optimized conditions, the proposed aptasensor showed a linear range of 0.1–20 nM with a detection limit of 34 pM. In addition, the proposed aptasensor had good stability and selectivity, offered promising choice for the detection of other small molecules.     

Target-Dependent Protection of DNA Aptamers against Nucleolytic Digestion Enables Signal-On Biosensing with Toehold-Mediated Rolling Circle Amplification

We report a generalizable strategy for biosensing that takes advantage of the resistance of DNA aptamers against nuclease digestion when bound with their targets, coupled with toehold mediated strand displacement (TMSD) and rolling circle amplification (RCA). A DNA aptamer containing a toehold extension at its 5′-end protects it from 3′-exonuclease digestion by phi29 DNA polymerase (phi29 DP) in a concentration-dependent manner. The protected aptamer can participate in RCA in the presence of a circular template that is designed to free the aptamer from its target via TMSD. The absence of the target leads to aptamer digestion, and thus no RCA product is produced, resulting in a turn-on sensor. Using two different DNA aptamers, we demonstrate rapid and quantitative real-time fluorescence detection of two human proteins: platelet-derived growth factor (PDGF) and thrombin. Sensitive detection of PDGF was also achieved in human serum and human plasma, demonstrating the selectivity of the assay.     

Metal-enhanced fluorescent probes based on silver nanoparticles and its application in IgE detection

In this paper, a novel metal plasmon coupled with an aptamer–nucleotide hybridized probe was fabricated and applied for protein detection. The specific aptamer and single-strand oligonucleotide were chemically bound to silver nanoparticles (AgNPs), and Cy5-labeled, complementary single-strand oligonucleotides were hybridized with the particle-bound oligonucleotides. The hybridized DNA duplexes were regarded as rigid rods that separated the fluorophore Cy5 and the surface of AgNPs to reduce the competitive quenching. Using a model system comprising human immunoglobulin E (IgE) as the analyte and goat antihuman IgE as immobilized capture antibody on glass slides, we demonstrate that the detection performance of the synthetic probe was superior to the aptamer-based fluorescent probes. The results showed a good linear correlation for human IgE in the range from 10 ng/ml to 6.25 μg/ml. The detection limit obtained was 1 ng/ml, which was 50 times lower than that using Cy5 oligonucleotide/aptamer hybrid duplex (Probe2) due to the metal-enhanced fluorescence effect. This new strategy opens the possibility for the preparation of high-sensitivity detection probes based on metal nanoparticles.     

Fluorescent sensing ochratoxin A with single fluorophore-labeled aptamer

We explored a fluorescent strategy for sensing ochratoxin A (OTA) by using a single fluorophore-labeled aptamer for detection of OTA. This method relied on the change of the fluorescence intensity of the labeled dye induced by the specific binding of the fluorescent aptamer to OTA. Different fluorescein labeling sites of aptamers were screened, including the internal thymine bases, 3′-end, and 5′-end of the aptamer, and the effect of the labeling on the aptamer affinity was investigated. Some fluorophore-labeled aptamers showed a signal-on or signal-off response. With the fluorescent aptamer switch, simple, rapid, and selective sensing of OTA at nanomolar concentrations was achieved. OTA spiked in diluted red wine could be detected, showing the feasibility of the fluorescent aptamer for a complex matrix. This method shows potential for designing aptamer sensors for other targets.

Fluorescence aptasensor based on competitive-binding for human neutrophil elastase detection

To our knowledge, we report the first fluorescence aptasensor for detecting human neutrophil elastase (HNE) in homogeneous solution. The biosensor contains a short DNA scrambled sequence strand (SS) complementary to part of the aptamer sequence or the loop of molecular beacon (MB). The aptamer-HNE recognition event involves competition between the molecular beacon and loose HNE aptamer for the binding the short DNA strand. The new biosensor can detect as little as 0.34 nM of HNE, and the response is linear in the tested concentration range of 0.34-68 nM with the detection limit of 47 pM.     

Highly sensitive electrochemiluminescence “turn-on” aptamer sensor for lead(II) ion based on the formation of a G-quadruplex on a graphene and gold nanoparticles modified electrode

We have developed a “turn on” model of an electrochemiluminescence (ECL) based assay for lead ions. It is based on the formation of a G-quadruplex from an aptamer labeled with quantum dots (QDs) and placed on an electrode modified with of graphene and gold nanoparticles (AuNPs). A hairpin capture probe was labeled with a thiol group at the 5′-end and with an amino group at the 3′-end. It was then self-assembled on the electrode modified with graphene and AuNPs. In the absence of Pb(II), the amino tag on one end of the hairpin probe is close to the surface of the electrode and therefore unable to interact with the QDs because of steric hindrance. The ECL signal is quite weak in this case. If, however, Pb(II) is added, the stem-loop of the aptamer unfolds to form a G-quadruplex. The amino group at the 3′-end will become exposed and can covalently link to a carboxy group on the surface of the CdTe QDs. This leads to strong ECL. Its intensity increases (“turns on”) with the concentration of Pb(II). Such a “turn-on” method does not suffer from the drawbacks of “turn-off” methods. ECL intensity is linearly related to the concentration of Pb(II) in the 10 p mol·L^?1 to 1 n mol·L^?1 range, with a 3.8 p mol·L^?1 detection limit. The sensor exhibits very low detection limits, good selectivity, satisfying stability, and good repeatability.

L-Argininamide biosensor based on S1 nuclease hydrolysis signal amplification

A sensitive method is presented for the detection of L-argininamide. It is based on the amplification of the hydrolysis of S1 nuclease of single-stranded regions of an aptamer-target complex. The S1 nuclease, which is sequence-independent, is used to “recycle” target molecules, thus leading to strongly enhanced chemiluminescence (CL). L-Argininamide was chosen as model analyte. The DNA aptamer and its complementary DNA were labeled with the CL reagent N-(4-aminobutyl)-N-ethylisoluminol (ABEI). The DNA complementary to the aptamer was labeled with ABEI and immobilized on magnetic beads (MBs) coated with gold. The aptamer was also labeled with ABEI and self-assembled on the MBs. A duplex was formed due to hybridization between the DNA aptamer and the DNA complementary to the aptamer. In the presence of the target L-argininamide, a stem-loop aptamer structure is formed which subsequently denatures the duplex. This switch from a duplex structure to a stem-loop structure causes the formation of single-stranded regions both in the target-aptamer and in the single-stranded DNA on the MBs. The nuclease hydrolyzes the single-stranded regions and single-stranded DNA. Ultimately, L-argininamide is released which then interacts with another aptamer on the MB, thereby leading to one more L-argininamide. This autocatalytic cycle can generate substantial quantities of ABEI which then can be sensitively determined by the diperiodatonickelate-isoniazide reaction system. L-argininamide can be detected in the concentration range from 3.0?×?10^?4 to 3.0?×?10^?7 M, and the limit of detection is 1.0?×?10^?7 M.

Aptamer based test stripe for ultrasensitive detection of mercury(II) using a phenylene-ethynylene reagent on nanoporous silver as a chemiluminescence reagent

We describe a paper-based chemiluminescence (CL) test for the determination of mercury(II) ion. A single-stranded DNA aptamer was first covalently immobilized via its amino groups to the hydroxy groups on the surface of cellulosic paper. The aptamer probes can capture Hg(II) ions due to their specific interaction with thymine. The CL reagent (a caboxylated phenylene-ethynylene referred to as P-acid) was immobilized on nanoporous silver (NPS@P-acid) and used a CL label on the aptamer. The stripe is then contacted with a sample containing Hg(II) ions and CL is induced by the addition of permanganate. CL intensity depends on the concentration of Hg(II) because Hg(II) increases the quantity of the P-acid-conjugated aptamer. The highly active surface of the NPS@P-acid composites results in an 8-fold higher CL intensity compared to the use of pure P-acid. This enables Hg(II) ion to be quantified in the 20 nM to 0.5 μM concentration range, with a limit of detection as low as 1 pM. This CL aptasensor is deemed to represent a promising tool for simple, rapid, and sensitive detection of Hg(II).

A pH-responsive activatable aptamer probe for targeted cancer imaging based on i-motif-driven conformation alteration

We present here a p H-responsive activatable aptamer probe for targeted cancer imaging based on i-motif-driven conformation alteration. This p H-responsive activatable aptamer probe is composed of two single-stranded DNA. One was used for target recognition, containing a central, target specific aptamer sequence at the 3′-end and an extension sequence at the 5′-end with 5-carboxytetramethylrhodamine(TAMRA) label(denoted as strand A). The other(strand I), being competent to work on the formation of i-motif structure, contained four stretches of the cytosine(C) rich domain and was labeled with a Black Hole Quencher 2(BHQ2) at the 3′-end. At neutral or slightly alkaline p H, strand I was hybridized to the extension sequence of strand A to form a double-stranded DNA probe, termed i-motif-based activatable aptamer probe(I-AAP). Because of proximityinduced energy transfer, the I-AAP was in a "signal off" state. The slightly acidic p H enforced the strand I to form an intramolecular i-motif and then initiated the dehybridization of I-AAP, leading to fluorescence readout in the target recognition. As a demonstration, AS1411 aptamer was used for MCF-7 cells imaging. It was displayed that the I-AAP could be carried out for target cancer cells imaging after being activated in slightly acidic environment. The applicability of I-AAP for tumor tissues imaging has been also investigated by using the isolated MCF-7 tumor tissues. These results implied the I-AAP strategy is promising as a novel approach for cancer imaging.     

Engineering a DNA-cleaving DNAzyme and PCR into a simple sensor for zinc ion detection

The development of a simple sensor (9NL27-Zn) based on DNAzyme and PCR and aimed at the detection of low concentrations of zinc (II) ions is described. A specific Zn(II)-dependent DNAzyme (9NL27) with DNA-cleaving activity was employed. In the presence of zinc (II), the DNAzyme hydrolyzed DNA substrate into two pieces (5′ and 3′ fragments), forming 3′-terminal hydroxyl in the 5′ fragment and 5′-phosphate in the 3′ fragments. Subsequently, the 5′ fragment left the DNAzyme and bound a short DNA template. The 5′ fragment was used as a primer and extended a single-stranded full-length template by Taq polymerase. Finally, this full-length template was amplified by PCR. The amplified products had a quantitative relationship with Zn(II) concentration. Under our experimental conditions, the DNA sensor showed sensitivity (10 nM) and high specificity for zinc ion detection. After improvement of the DNA sensor, the detection limit can reach 1 nM. The simple DNA sensor may become a DNA model for the detection of trace amounts of other targets.

Determination of Enrofloxacin in Bovine Milk by a Novel Single-Stranded DNA Aptamer Chemiluminescent Enzyme Immunoassay

Enrofloxacin, a widely used fluoroquinolone antibiotic, may be a cause of bacterial drug resistance and is forbidden in poultry. Consequently, a sensitive and rapid method is required for its determination. Aptamers, which are more stable and easily synthesized than antibodies, may serve as alternatives in the development of methods for rapid detection. Six single-strand DNA aptamers binding to enrofloxacin were selected by in vitro selection. Aptamer number 17 showed the highest affinity for enrofloxacin with a dissociation constant of 188 nM and the highest guanine concentration (35%), which was predicted to be crucial for strong affinity of the aptamer to enrofloxacin, and successfully distinguished enrofloxacin from its structure analogs. Using aptamer number 17, a novel chemiluminescent enzyme immunoassay associating with biotin-streptavidin was developed that allowed the determination of enrofloxacin to 2.26 ng/mL. Due to its capability to determine enrofloxacin in bovine milk, this newly selected aptamer may find broad application in food and environmental monitoring.     

A fluorometric aptasensor for methamphetamine based on fluorescence resonance energy transfer using cobalt oxyhydroxide nanosheets and carbon dots

Cobalt oxyhydroxide (CoOOH) nanosheets are efficient fluorescence quenchers due to their specific optical properties and high surface area. The combination of CoOOH nanosheets and carbon dots (CDs) has not been used in any aptasensor based on fluorescence quenching so far. An aptamer based fluorometric assay is introduced that is making use of fluorescent CDs conjugated to the aptamer against methamphetamine (MTA), and of CoOOH nanosheets which reduce the fluorescence of the CDs as a quencher. The results revealed that the conjugated CDs with aptamers were able to enclose the CoOOH nanosheets. Consequently, fluorescence is quenched. If the aptamer on the CD binds MTA, the CDs are detached from CoOOH nanosheets. As a result, fluorescence is restored proportionally to zhe MTA concentration. The fluorometric limit of detection is 1 nM with a dynamic range from 5 to 156 nM. The method was validated by comparing the results obtained by the new method to those obtained by ion mobility spectroscopy. Theoretical studies showed that the distance between CoOOH nanosheet and C-Ds is approximately 7.6 Å which can illustrate the possibility of FRET phenomenon. The interactions of MTA and the aptamer were investigated using molecular dynamic simulation (MDS).

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Graphical abstract Carbon dots (C-Ds) were prepared from grape leaves, conjugated to aptamer, and adsorbed on CoOOH nanosheets. So, the fluorescence of C-Ds is quenched. On addition of MTA, fluorescence is restored.

     

Aptamer based ultrasensitive determination of the β-adrenergic agonist ractopamine using PicoGreen as a fluorescent DNA probe

The authors describe an aptamer based assay for determination of ractopamine (RAC) by using PicoGreen (PG) as a fluorescent probe specific for dsDNA. In the absence of RAC, the aptamer forms a duplex structure with a complementary sequence that results in enhanced PG fluorescence. Upon binding to RAC, the aptamer undergoes a structural switch. This reduces the number of DNA duplexes formed and causes a reduction of fluorescence intensity of PG as measured at excitation/emission wavelengths of 480/520 nm. Under optimized conditions, the dynamic calibration plot covers the 50 pM to 50 μM concentration range, with a 50 pM detection limit. This meets the safety supervision regulations of the European Commission in terms of residue limits of RAC in food. The method displays high selectivity over other β-adrenergic agonists including clenbuterol, dopamine and salbutamol. The assay was successfully applied to samples of swine urine at spiking levels of 7.4 nM, 22.2 nM and 37 nM. Average recoveries ranged from 95 to 110%, with an RSD of <1.5%. The method is expected to represent a promising tool for simple, rapid and sensitive on-site detection of RAC in animal products.

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Graphical abstract An aptamer based fluorescent assay for determination of ractopamine was developed with a dynamic range of 50 pM to 50 μM. The average recovery from spiked urine samples ranged from 95 to 110%, with an RSD of <1.5%.

     

Fluorescent assay for oxytetracycline based on a long-chain aptamer assembled onto reduced graphene oxide

We report on a fluorescent assay for oxytetracycline (OTC) using a fluorescein-labeled long-chain aptamer assembled onto reduced graphene oxide (rGO). The π-π stacking interaction between aptamer and rGO causes the fluorescence of the label to be almost completely quenched via energy transfer so that the system has very low background fluorescence. The addition of OTC leads to the formation of G-quadruplex OTC complexes and prevents the adsorption of labeled aptamer on the surface of rGO. As a result, fluorescence is restored, and this effect allows for a quantitative assay of OTC over the 0.1–2 μM concentration range and with a detection limit of 10 nM. This method is simple, rapid, selective and sensitive. It may be applied to other small molecule analytes by applying appropriate aptamers.

Colorimetric aptamer based assay for the determination of fluoroquinolones by triggering the reduction-catalyzing activity of gold nanoparticles

The authors describe a colorimetric assay for the detection of fluoroquinolones (FQs). It is based on the use of gold nanoparticles (AuNPs) modified with complementary DNA strands and analyte-specific FQ-binding aptamers. The modified AuNPs possess enzyme-like activity that can catalyze the reduction of nitrophenol by NaBH₄. In the absence of ciprofloxacin, the flower-shape coating on the AuNPs prevents the reduction of yellow 4-nitrophenol. In the presence of ciprofloxacin, the DNA/aptamer flower leaves on the AuNPs and the AuNPs can exert their catalytic activity. This results in a color change from yellow to colorless. The assay is highly selective for FQs, fast (1 h), and has a limit of detection as low as 1.2 nM in case of ciprofloxacin. It was successfully applied to the determination of ciprofloxacin in spiked water, serum and milk samples to give LODs of 1.3, 2.6 and 3.2 nM, respectively.     

A selective adenosine sensor derived from a triplex DNA aptamer

The aim of this study is to develop a selective adenosine aptamer sensor using a rational approach. Unlike traditional RNA aptamers developed from SELEX, duplex DNA containing an abasic site can function as a general scaffold to rationally design aptamers for small aromatic molecules. We discovered that abasic site-containing triplex DNA can also function as an aptamer and provide better affinity than duplex DNA aptamers. A novel adenosine aptamer sensor was designed using such a triplex. The aptamer is modified with furano-dU in the binding site to sense the binding. The sensor bound adenosine has a dissociation constant of 400 nM, more than tenfold stronger than the adenosine aptamer developed from SELEX. The binding quenched furano-dU fluorescence by 40%. It was also demonstrated in this study that this sensor is selective for adenosine over uridine, cytidine, guanosine, ATP, and AMP. The detection limit of this sensor is about 50 nM. The sensor can be used to quantify adenosine concentrations between 50 nM and 2 μM.     

A novel fluorescent aptasensor based on silica nanoparticles,PicoGreen and exonuclease III as a signal amplification method for ultrasensitive detection of myoglobin

Measurement of myoglobin (Mb) in human blood serum is of great interest for quick diagnosis of acute myocardial infarction (AMI). In this study, a novel fluorescent aptasensor was designed for ultrasensitive and selective detection of Mb, based on target-induced high fluorescence intensity, complementary strand of aptamer (CS), PicoGreen (PG) dye, exonuclease III (Exo III) and silica nanoparticles coated with streptavidin (SNPs-Streptavidin). The developed aptasensor obtains characteristics of SNPs as enhancers of fluorescence intensity, Exo III as an enzyme which selectively digests the 3'-end of double-stranded DNA (dsDNA), PG as a fluorescent dye which could selectively bind to dsDNA and high selectivity and sensitivity of aptamer (Apt) toward its target. In the absence of Mb, no free CS remains in the environment of SNPs-Streptavidin, resulting in a weak fluorescence emission. In the present of Mb, dsDNA-modified SNPs-Streptavidin complex forms, leading to a very strong fluorescence emission. The developed fluorescent aptasensor exhibited high specificity toward Mb with a limit of detection (LOD) as low as 52 pM. In addition, the designed fluorescent aptasensor was efficiently used to detect Mb in human serum.     

A homogeneous and “off–on” fluorescence aptamer-based assay for chloramphenicol using vesicle quantum dot-gold colloid composite probes

In this work, a novel homogeneous and signal “off–on” aptamer based fluorescence assay was successfully developed to detect chloramphenicol (CAP) residues in food based on the fluorescence resonance energy transfer (FRET). The vesicle nanotracer was prepared through labeling single stranded DNA binding protein (SSB) on limposome-CdSe/ZnS quantum dot (SSB/L-QD) complexes. It was worth mentioning that the signal tracer (SSB/L-QD) with vesicle shape, which was fabricated being encapsulated with a number of quantum dots and SSB. The nanotracer has excellent signal amplification effects. The vesicle composite probe was formed by combining aptamer labeled nano-gold (Au-Apt) and SSB/L-QD. Which based on SSB's specific affinity towards aptamer. This probe can't emit fluoresce which is in “off” state because the signal from SSB/L-QD as donor can be quenched by the Au-aptas acceptor. When CAP was added in the composite probe solution, the aptamer on the Au-Apt can be preferentially bounded with CAP then release from the composite probe, which can turn the “off” signal of SSB/L-QD tracer into “on” state. The assay indicates excellent linear response to CAP from 0.001 nM to 10 nM and detection limit down to 0.3 pM. The vesicle probes with size of 88 nm have strong signal amplification. Because a larger number of QDs can be labeled inside the double phosphorus lipid membrane. Besides, it was employed to detect CAP residues in the milk samples with results being agreed well with those from ELISA, verifying its accuracy and reliability.     

A Novel Fluorescence Sensor for Cocaine with Signal Amplification through Cycling Exo-Cleaving with a Hairpin Probe

A novel fluorescence assay, based on the signal amplification through cycling reactions of aptameric recognition and nucleic acid exo-cleaving, was established for the sensitive detection of cocaine. In this assay, a new single fluorophore-labeled DNA-hairpin probe was used. This smart probe was established based on the significant nucleobase quenching between the nucleobases and the fluorophore. In the presence of cocaine and its aptamer, the structure of the smart probe changed to recover the fluorescence. In order to enhance the sensitivity of this assay, exonuclease III was introduced to enable the inputted cocaine to react with multiple probes in a recycling manner. Under the optimal conditions, a linear range for cocaine from 4.0 × 10^?9 to 8.0 × 10^?8 M with a detection limit of 1.76 × 10^?9 M (3σ, n = 11) was obtained.