An ultrasensitive electrochemical detection of target DNA was developed based on target-triggered hairpin assembly and exonuclease III (Exo III)-assisted recycling quadratic amplification strategy. The detection employed a gold nanoparticles (AuNPs) modified Au electrode and two specially designed hairpin probes P1 and P2. P1 probe contained G-quadruplex-forming sequence and target DNA recognition region, and was immobilized on the electrode. P2 probe was used as a secondary complementary sequence which can displace target DNA and hybridize with P1 probe. In the absence of target DNA, these hairpin structures of P1 and P2 can coexist. While in the presence of target DNA, it can trigger the self-assembly process of P1 and P2 and initiate the Exo III-assisted two recycling process, resulting in the formation of G-quadruplex structure on electrode surface. Finally, with the addition of hemin, numerous G-quadruplex-hemin complexes formed on the electrode surface and gave a pronounced electrochemical response in differential pulse voltammogram (DPV). Taking K-ras proto oncogene as an example, the proposed DNA biosensor exhibited a wide detection range from 10 fM to 20 nM, and an extremely low detection limit of 2.86 fM. Moreover, it can clearly discriminate one-base difference in DNA sequence, thus can identify the mutation of the target gene. The proposed DNA biosensor has potential applications in the fields of clinic diagnosis, biomedicine, food and environment microbial monitoring.Detection of target DNA was based on target-triggered hairpin assembly and exonuclease III (Exo III)-assisted recycling quadratic amplification strategy.Download high-res image (152KB)Download full-size image

A UV-visible spectroscopic detection method of kanamycin was successfully developed based on target-induced growth of gold nanoparticles (AuNPs), using AuNPs as the probe and a kanamycin-specific aptamer as the recognition element. Firstly, the aptamer was incubated with AuNPs, which blocked the surface of the AuNPs, and inhibited the further growth of AuNPs. However, kanamycin bound with the aptamer on the surface of AuNPs with high affinity, competitively desorbed the aptamer and exposed the surface of AuNPs. Then, the growth of AuNPs proceeded by addition of NH2OH and HAuCl4. After the growth of AuNPs, the color and the maximum absorption wavelength of the AuNP solution changed, which was utilized to determine the concentration of kanamycin. Under the optimized conditions, the proposed UV-visible spectroscopic assay showed high sensitivity and high specificity for kanamycin, with a linear detection range from 20 to 100 nM, and the detection limit of 9.21 nM. Moreover, the assay has been successfully applied to detect kanamycin in honey samples without pretreatment. Therefore, it has great application prospects to detect residual kanamycin in food, medicine and water environments.

•A direct electrochemical assay for kanamycin was presented based on nanozyme.•The assay has extremely high sensitivity with the detection limit down to 60 pM.•The assay has great prospect in detection of harmful substances in food samples.An enzyme-free, ultrasensitive electrochemical detection of kanamycin residue was achieved based on mimetic peroxidase activity of gold nanoparticles (AuNPs) and target-induced replacement of the aptamer. AuNPs which were synthesized using tyrosine as a reducing and capping agent, exhibited mimetic peroxidase activity. In the presence of kanamycin-specific aptamer, however, the single-stranded DNA (ssDNA) adsorbed on the surface of AuNPs via the interaction between the bases of ssDNA and AuNPs, and therefore blocked the catalytic site of AuNPs, and inhibited their peroxidase activity. While in the presence of target kanamycin, it bound with the adsorbed aptamer on AuNPs with high affinity, exposed the surface of AuNPs and recovered the peroxidase activity. Then AuNPs catalyzed the reaction between H2O2 and reduced thionine to produce oxidized thionine. The latter exhibited a distinct reduction peak on gold electrode in differential pulse voltammetry (DPV), and could be utilized to quantify the concentration of kanamycin. Under the optimized conditions, the proposed electrochemical assay showed an extremely high sensitivity towards kanamycin, with a linear relationship between the peak current and the concentration of kanamycin in the range of 0.1–60 nM, and a detection limit of 0.06 nM. Moreover, the established approach was successfully applied in the detection of kanamycin in honey samples. Therefore, the proposed electrochemical assay has great potential in the fields of food quality control and environmental monitoring.

A sensitive simultaneous detection of streptomycin (STR), chloramphenicol (CHL) and tetracycline (TET) residues was achieved based on aptamers and quantum dot (QD) tags. Three complementary DNA1 sequences (cDNA1s) were first designed, which are not only complementary to corresponding aptamer DNAs (Ap-DNAs) specific for STR, CHL and TET, but also complementary to part of capture DNAs (Cap-DNAs) and part of complementary DNA2s (cDNA2s), respectively. cDNA1s initially hybridize with corresponding Ap-DNAs to form duplex DNAs. However, when the target antibiotics are present, STR, CHL and TET can bind with their Ap-DNAs specifically with higher affinity, which leads to the release of cDNA1s. Then the liberated cDNA1s hybridize with corresponding Cap-DNAs which are immobilized on the surface of a gold electrode by self-assembly. After that, the prepared PbS, CdS and ZnS QDs-tagged cDNA2s further hybridize with the other ends of corresponding cDNA1s on the electrode surface. The captured QDs yield distinct electrochemical signals after acid dissolution, which reflect the type and concentration of target antibiotics. Due to the inherent amplification feature of QD tags, the low detection limits for STR, CHL and TET reached 10 nM, 5 nM and 20 nM, respectively. In addition, the established assay was applied in the detection of STR, CHL and TET residues in milk samples and showed similar response ranges.

A novel strategy was established for simultaneous electrochemical detection of dual target DNAs. A template DNA was firstly designed to be complementary to target DNA 1, target DNA 2, and part of signal DNA, which contains a G-quadruplex forming sequence. In the presence of both target DNAs and signal DNA, these sequences hybridized with template DNA and were further ligated together to form a long strand under the catalysis of E. coli DNA ligase. After denaturation, the ligated sequence was dehybridized with template DNA and captured with a capture probe immobilized on the surface of a gold electrode. With the help of hemin, a G-quadrupelx–hemin complex can be formed on the surface of the gold electrode which produced a remarkable electrochemical signal. Therefore, the proposed DNA sensor was used to simultaneously detect dual target DNAs with extremely high sensitivity. For target DNA 1 and target DNA 2, detection limits of 100 fM and 7.4 fM were achieved respectively. The specificity of the sensor was verified by employing one-base mismatched and three-base mismatched sequences. In consideration of its potential for multiplex DNA sequence detection, the proposed DNA sensor may have great potential in the fields of microbial identification, disease diagnosis, food quality control and environmental monitoring.

Kanamycin is a widely used aminoglycoside antibiotic, and residual kanamycin in animal-derived food causes serious side effects. Herein, a simple aptamer-based assay is proposed for the label-free electrochemical detection of kanamycin. A 5′-SH-modified kanamycin-specific aptamer was self-assembled on the surface of a gold electrode through Au–S chemistry. Upon binding to kanamycin, the conformation of the aptamer changes and its coverage of the electrode surface increases, owing to the formation of a stem-loop structure in the complex. As a result, the electron transfer resistance between the solution species and the electrode is increased. Using [Fe(CN)6]3−/4− as a probe, the square wave voltammetry (SWV) current response was utilised to determine the concentration of kanamycin. The detection range of the aptasensor was found to be 10–2000 nM, indicating a high sensitivity and a broad detection range. The specificity of detection was also determined. Furthermore, the assay was successfully employed in the detection of kanamycin in milk samples, with a similar response range and detection limit.

•A complex electrochemical assay couples the detection of target DNAs with LDH.•A positive response can only be gained in the presence of both target DNAs and LDH.•The sensor detects dual-target DNA with broad range and low detection limit.We report here an electrochemical sensor which couples the detection of target DNAs with enzyme activity. A hairpin probe DNA complementary to both target DNAs was dually labeled with 5′-thiol and 3′-methylene blue (MB), and covalently immobilized to gold electrode. In the presence of both target DNAs, the hairpin structure shifted from “close” state to “open” state with the help of Escherichia coli (E. coli) DNA ligase, which can be characterized by alternating current voltammetry (ACV). For target DNA 1 and target DNA 2, the detection limits were 1 nM and 0.25 nM, respectively. Furthermore, the assay was coupled with the activity of lactate dehydrogenase (LDH), which catalyzes the conversion of NADH to NAD+. The latter acted as the co-substrate of DNA ligase and produced current change in the presence of both target DNAs. The sensor can analyze the co-existence of multi-components, which efficiently improves the accuracy in the applications such as species identification.

Aminoglycoside antibiotics are widely used drugs. The residual antibiotics in animal-derived food are proven to be harmful to human health and therefore should be strictly controlled in the food industry. A spectrophotometric method based on a kanamycin-specific aptamer and gold nanoparticles (AuNPs) was developed for the quantitative detection of kanamycin. Two types of functionalized AuNPs were synthesized by self-assembly of thiol-modified single-stranded DNAs (ssDNAs), which were complementary to the 5′ terminal and 3′ terminal sequences of the kanamycin aptamer. As the kanamycin aptamer is mixed with these AuNPs, the AuNPs aggregate due to the hybridization of the aptamer with the complementary ssDNAs on the surface of AuNPs. However, when different concentrations of kanamycin are present, it can bind with the aptamer specifically and competitively, which leads to the disaggregation of the AuNPs aggregate, and a change in the UV-visible absorption spectrum of AuNPs. The absorbance at 527 nm is utilized to detect the concentration of kanamycin. The detection range of this method is 1–500 nM, with a detection limit of 1 nM. The developed method was then successfully employed to detect kanamycin in milk samples, and a similar response range and detection limit were obtained.

Protein–DNA interaction plays important roles in many cellular processes, and there is an urgent demand for valid methods to monitor the interaction. In view of this, we propose a simple label-free colorimetric platform for the detection of protein–DNA interaction. Protein–DNA couples together with peroxidase-mimicking DNAzyme and exonuclease are elaborately incorporated into an integrated biosensing system. Besides the simplicity and efficiency, the strategy also has a great advantage for its universality in the detection of different protein–DNA couples. In our experiments, effective validation of our approach can be supported by two different protein–DNA couples (estrogen receptor α and nuclear factor kappa B). Experimental results show that the DNAzyme is competent to give rise to evident readout signals to monitor protein–DNA couples. Furthermore, with the substitution of DNA binding sequence in the probe, this method could be extended to a general platform for the detection of protein–DNA interaction.Highlights► A novel colorimetric biosensing platform for the detection of protein–DNA interaction. ► Interaction between ERα/NF-κB and their corresponding binding sequences is studied. ► The biosensing platform is easily-operated, cost-effective and universal.

Single-stranded DNA (ssDNA) aptamers specific to streptomycin were screened and identified from a random oligonucleotides library by affinity magnetic beads-based SELEX. After eight rounds of selection, 16 ssDNA with different sequences were identified. Then the dissociation constants (Kd) of these ssDNA were determined and an aptamer (STR1) with highest affinity for streptomycin was identified. Further study showed that aptamer STR1 exhibits very low affinity for other aminoglycoside antibiotics, indicating high specificity. With this aptamer, detection of streptomycin was achieved by using gold nanoparticles (AuNPs)-based colorimetric method. In the presence of streptomycin, the competitive binding of the target and the aptamer decreases the stability of AuNPs in NaCl solution, triggers the aggregation, and exhibits visible color change of AuNPs solution. Through UV–visible spectroscopic quantitative analysis, streptomycin can be detected in the range of 0.2–1.2 μM. The presence of other aminoglycoside antibiotics shows neglectable disturbance. Furthermore, the established method was utilized to detect streptomycin in honey, and the same low detection limit and linear detection range were achieved.Highlights► Sixteen ssDNA aptamers specific to streptomycin were screened and identified. ► The structures and the dissociation constants of the aptamers were characterized. ► STR1 was used in the detection of streptomycin in both solution and honey samples. ► The sensitivity, specificity and repeatability of the assay were studied.

A spectrophotometric method for the detection of tyrosinase activity is developed by utilizing the product-triggered aggregation of boronic acid-functionalized gold nanoparticles. Based on the changes of absorbance in UV-visible spectra, the assay shows extremely high sensitivity and lowered limit of detection of 1 × 10−10 u mL−1.

Aminoglycoside antibiotics are widely used drugs. The residual antibiotics in animal-derived food are proven to be harmful to human health and therefore should be strictly controlled in the food industry. A spectrophotometric method based on a kanamycin-specific aptamer and gold nanoparticles (AuNPs) was developed for the quantitative detection of kanamycin. Two types of functionalized AuNPs were synthesized by self-assembly of thiol-modified single-stranded DNAs (ssDNAs), which were complementary to the 5′ terminal and 3′ terminal sequences of the kanamycin aptamer. As the kanamycin aptamer is mixed with these AuNPs, the AuNPs aggregate due to the hybridization of the aptamer with the complementary ssDNAs on the surface of AuNPs. However, when different concentrations of kanamycin are present, it can bind with the aptamer specifically and competitively, which leads to the disaggregation of the AuNPs aggregate, and a change in the UV-visible absorption spectrum of AuNPs. The absorbance at 527 nm is utilized to detect the concentration of kanamycin. The detection range of this method is 1–500 nM, with a detection limit of 1 nM. The developed method was then successfully employed to detect kanamycin in milk samples, and a similar response range and detection limit were obtained.