An amperometric aptasensor is reported for the electrochemical determination of the epithelial cell adhesion molecule (EpCAM). It is based on a combination of EpCAM-driven toehold-mediated DNA recycling amplification, the specific recognition of EpCAM aptamer, and its binding to EpCAM. Hairpin probe 1 (Hp1) with a toehold region was modified with a 5′-thiol group (5’-SH) and self-assembled onto the surface of a gold electrode. Upon addition of EpCAM, the probe A (a 15-mer) is liberated from the aptamer/probe A complex and then hybridizes with the toehold domain of Hp1. This results in the exposure of another toehold for further hybridizing with hairpin probe 2 (Hp2) to displace probe A in the presence of Hp2 that was labeled with the electrochemical probe Methylene Blue (MB). Subsequently, liberated probe A is hybridized again with another Hp1 to start the next round of DNA recycling amplification by reusing probe A. This leads to the formation of plenty of MB-labeled DNA strands on the electrode surface and generates an amplified current. This 1:N probe-response amplification results in ultrasensitive and specific detection of EpCAM, with a 20 pg·mL?1 detection limit. The electrode is highly stable and regenerable. It was successfully applied to the determination of EpCAM in spiked human serum, urine and saliva, and thus provides a promising tool for early clinical diagnosis.
Graphical abstract Schematic illustration of the electrochemical detection for EpCAM. The method is based on aptamer-based recognition and EpCAM-driven toehold-mediated DNA recycling amplification. Hp1: Hairpin probe 1; Hp2: Hairpin probe 2; MB: Methylene blue; MCH: 6-Mercapto-1-hexanol; EpCAM: Epithelial cell adhesion molecule.
An electrochemical biosensor for determination of DNA is described that is based on the reaction of regulated DNA (reg-DNA) first with substrated DNA (subs-DNA) to form a reaction intermediate. The intermediate binds target DNA (T) by hybridization and initiates a branch migration leading to the production of complex of substrated DNA and target DNA (TC). Once TC is produced, it reacts with assisted DNA (ass-DNA) through a toehold exchange mechanism, yielding the product complex of substrated DNA and assisted DNA (CS). The target is then released back into the solution and and catalyzes the next cycle of toehold-exchange with the reaction intermediate of substrated DNA and regulated DNA (CPR). Unlike in a conventional DNA toehold that is hardwired with the branch migration domain, the allosteric DNA toehold is designed into a reg-DNA which is independent of the branch migration domain. Under the optimal experimental conditions and at a working potential as low as 0.18 V, response to DNA is linear in the 1 fM to 1000 pM concentration range, and the detection limit is 0.83 fM. The assay is highly specific and can discriminate target DNA even from a single-base mismatch. It was applied to the analysis of DNA spiked plasma samples.
Graphical abstract Schematic illustration of the electrochemical strategy for target DNA detection based on regulation of DNA strand displacement using an allosteric DNA toehold strategy. It can be used to analyze DNA-spiked plasma samples and has a low detection limit of 0.83 fM.
MicroRNAs (miRNAs) play a considerable role in cancer occurrence and development, and have been identified as promising noninvasive biomarkers. The authors describe a voltammetric method for the determination of the cancer biomarker microRNA-21 (miRNA). It is based on a combination of a universal DNA signal transducer and isothermal target recycling amplification. A hairpin capture probe is bound to the target miRNA to form a duplex structure and to create a toehold in the transducer for initiating the target recycling amplification reaction. In contrast to traditional capture probes, a mismatched site is introduced to improve its ability to capture the target. In order to reduce the complex design procedures of the sequence and widen the applicability of this method, a signal transducer is introduced. Under optimal conditions, response to target miRNA is linear in the 0.5 to 2000 pM concentration range, with a 56 fM. detection limit (at an S/N ratio of 3). In order to characterize the process of target recycling and the stepwise modification of the electrode, real-time fluorescence, agarose gel electrophoresis, cyclic voltammetry, electrochemical impedance spectroscopy and chronocoulometry were used. The results indicate that this isothermal target recycling amplification results in an electrochemical biosensing scheme with wide potential for sensing other bioanalytes.
Graphical abstract Schematic illustration of the electrochemical biosensing platform for miRNA-21 detection based on isothermal target recycling amplification and DNA signal transducer triggered strategy.
The authors describe a colorimetric method for the determination of Hg(II) ion. It is based on the color change from red to colorless as displayed by gold nanoparticle (AuNP) modified with thymine - rich DNA. Signal amplification is accomplished by free strand displacement recycling. In this strategy, Hg(II) unfolds the arch-trigger duplex due to the high affinity between Hg(II) and the thymines to form T-Hg(II)-T structures, thereby causing the release of trigger. The liberated trigger unfolds the hairpin structure of H1, and unfolded H1 further unfolds with H2. As a result, the H2 hairpin displaces trigger, and the released trigger unfolds another H1. This results in strong and enzyme-free strand displacement recycling amplification. The aggregation of DNA-AuNPs occurs in the presence of the duplex formed by hairpins H2 and H1. This results in a color change from red to colorless that can be visually observed. Under optimal conditions, the assay has a detection range over 4 orders of magnitude and a 3.4 nM detection limit. The assay is selective, sensitive, rapid and cost-effective. In our perception, it represents a useful platform for determination of Hg(II).
Graphical abstract Schematic presentation of the simple, rapid, low cost colorimetric detection of mercury(II) based on enzyme-free strand displacement amplification along with DNA-labeled AuNP.
An isothermal colorimetric method is described for amplified detection of the CaMV 35S promoter sequence in genetically modified organism (GMO). It is based on (a) target DNA-triggered unlabeled molecular beacon (UMB) termini binding, and (b) exonuclease III (Exo III)-assisted target recycling, and (c) hemin/G-quadruplex (DNAzyme) based signal amplification. The specific binding of target to the G-quadruplex sequence-locked UMB triggers the digestion of Exo III. This, in turn, releases an active G-quadruplex segment and target DNA for successive hybridization and cleavage. The Exo III impellent recycling of targets produces numerous G-quadruplex sequences. These further associate with hemin to form DNAzymes and hence will catalyze H2O2-mediated oxidation of the chromogenic enzyme substrate ABTS2? causing the formation of a green colored product. This finding enables a sensitive colorimetric determination of GMO DNA (at an analytical wavelength of 420 nm) at concentrations as low as 0.23 nM. By taking advantage of isothermal incubation, this method does not require sophisticated equipment or complicated syntheses. Analyses can be performed within 90 min. The method also discriminates single base mismatches. In our perception, it has a wide scope in that it may be applied to the detection of many other GMOs.
Graphical abstract An isothermal and sensitive colorimetric method is described for amplified detection of CaMV 35S promoter sequence in genetically modified organism (GMO). It is based on target DNA-triggered molecular beacon (UMB) termini-binding and exonuclease III assisted target recycling, and on hemin/G-quadruplex (DNAzyme) signal amplification.
The authors describe a pipette type of biosensor for detecting target genes and using a zinc finger protein fused to luciferase (ZF luciferase). The ZF protein binds to a specific DNA sequence, and the target double-stranded (ds) DNA can be detected by monitoring the enzymatic activity of ZF luciferase. A small avidin-immobilized reaction plate is placed on a plastic pipette tip (referred to as Biologi tip). The dsDNA detection procedures are carried out by using a programmable dispensing robot equipped with a photodetector. These procedures include (a) the aspiration of an analyte to capture the biotinylated target dsDNA (a product of a polymerase chain reaction) on the small reaction plate inside the pipette tip, (b) the introduction of ZF luciferase and luciferin into the pipette tip, and (c) migration of the pipette tip to the detection port to measure bioluminescence on the small reaction plate. The emission originating from luciferase activity is observed on the reaction plate containing immobilized biotin-tagged target dsDNA, whereas plates containing non-target or biotinylated single-stranded DNA only do not yield a signal. The intensity of emission increases proportionally to the concentration of dsDNA, and the detection limit of the target dsDNA is as low as 62 pM. An actual genomic DNA sample from Escherichia coli O157 was successfully detected by this automatic analyzer using the Biologi tip equipped with a reaction plate. This indicates that this system has a large potential for practical applications, including in particular point-of-care analyses in hygiene control, food safety testing, and clinical diagnosis.
Graphical abstract A pipette-type biosensor was developed to detect target genes using a luciferase-fused zinc finger protein, where a small NeutrAvidin-immobilized reaction plate was placed on the tip, and the biotinylated target double-stranded DNA was detected by monitoring the bound luciferase activity.
The authors report on a simple strategy for sensitive determination of the activity of terminal deoxynucleotidyl transferase (TdT) using copper nanoclusters (CuNCs) as fluorescent probes. TdT-polymerized long chain AT-rich DNA serves as a template for the synthesis of the CuNCs, and TdT activity is detected fluorometrically at excitation/emission wavelengths of 340/570 nm. The protocol relies on the target-triggered formation of dsDNA polymers and in-situ formation of CuNCs. The calibration plot is linear in the 0.7 to 14 U L?1 activity range, with a 60 mU L?1 detection limit (at a signal-to-noise ratio of 3). The protocol was applied to determine TdT activity in acute lymphatic leukemia cells. This approach is selective, simple, convenient and cost-efficient because a complex DNA sequence is not required. In our perception, the method provides a viable new platform for monitoring the activity and inhibition of TdT.
Graphical abstract Based on the target-triggered formation of dsDNA polymers and in-situ formation of CuNCs with strong fluorescence, a turn-on fluorescence assay for TdT activity is presented.
A colorimetric assay is described for the detection of BCR/ABL fusion genes. Polyamidoamine (PAMAM) dendrimers were placed on peroxidase (POx) mimicking Au@Pt nanoparticles to form a nanocomposite of type Au@Pt-PAMAM. Capture DNA probe is a designed nucleic acid strand that specifically binds target DNA to the surface of the electrode. The capture probe was attached to magnetic beads via biotin and avidin interaction. The hairpin structure of the capture probe can only be opened by the complementary BCR/ABL DNA. This results in a highly specific assay. The POx-mimicking property of the Au@Pt-PAMAM causes the formation of a blue dye by reaction of H2O2 and 3,3,3′,3′-tetramethylbenzidine (TMB) which is measured by a microplate reader. Under optimum conditions, the absorbance increases linearly the 1 pM to 100 nM BCR/ABL concentration range, and the detection limit is as low as 190 fM. The method is highly selective and was successfully applied to the determination of fusion genes in spiked real samples. Conceivably, it possesses a large potential in clinical testing of patients suffering from chronic myeloid leukemia.
Graphical abstract Au@PtNP, an efficient catalyst, is bound with polyamidoamine (PAMAM) dendrimer to amplify the colorimetric signal. With the introduction of streptavidin-magnetic beads to remove non-specific signals, a novel colorimetric sensor is constructed to detect BCR/ABL fusion genes.
An aptamer based assay is described for the colorimetric detection of adenosine. The presence of adenosine triggers the deformation of hairpin DNA oligonucleotide (HP1) containing adenosine aptamer and then hybridizes another unlabeled hairpin DNA oligonucleotide (HP2). This leads to the formation of a double strand with a blunt 3′ terminal. After exonuclease III (Exo III)-assisted degradation, the guanine-rich strand (GRS) is released from HP2. Hence, the adenosine-HP1 complex is released to the solution where it can hybridize another HP2 and initiate many cycles of the digestion reaction with the assistance of Exo III. This leads to the generation of a large number of GRS strands after multiple cycles. The GRS stabilize the red AuNPs against aggregation in the presence of potassium ions. If, however, GRS forms a G-quadruplex, it loses its ability to protect gold nanoparticles (AuNPs) from salt-induced AuNP aggregation. Therefore, the color of the solution changes from red to blue which can be visually observed. This colorimetric assay has a 0.13 nM detection limit and a wide linear range that extends from 5 nM to 1 μM.
Graphical abstract Schematic presentation of a colorimetric aptamer biosensor for adenosine detection based on DNA cycling amplification and salt-induced aggregation of gold nanoparticles.
The authors describe an electrochemical strategy for highly sensitive determination of ATP that involves (a) aptamer-based target recognition, (b) enzyme-free dendritic DNA nanoassembly amplification with multiplex binding of the biotin-strepavidin system, and (c) enzyme-amplified differential pulse voltammetric readout. In the presence of ATP, binding of ATP to the aptamer releases trigger DNA from the double-stranded complex between ATP aptamer and trigger DNA. The single-stranded thiolated capture probe, chemisorbed on the gold electrode surface, captures the released trigger DNA via hybridization. The toehold of the trigger DNA is recombined with one end of the first substrate DNA (1) which is on its other end biotinylated and blocked, with loops, by a counterstrand. The latter is removed by a complementary single-stranded helper (1) exposing two toeholds and two identical complimentary sequences for a second biotinylated substrate DNA (2). The latter, which is double-stranded except for the toehold, binds to one of these two sites. It is then stripped from its counter strand by another single-stranded helper DNA 2, exposing a toehold to bind another substrate DNA 1. On this substrate, another cycle with dentrimeric bransching can start.Substrate 1 with its two binding sites for substrate 2 initiates the assembly of dendritic DNA on the surface of the gold electrode, which finally possesses numerous biotins at the terminal ends of both of the associated substrate DNAs. Subsequent multiplex binding of streptavidinylated alkaline phosphatase and enzyme-amplified electrochemical readout leads to a highly sensitive electrochemical ATP aptasensor. If operated in the DPV mode, the current as measured at a typical working potential of 0.25 V (vs. Ag/AgCl) increases linearly over the 10 nM to 10 μM logarithmic ATP concentration range, and the detection limit is 5.8 nM (at an S/N ratio of 3). The assay is highly specific and reproducible. It was successfully applied to the detection of ATP in spiked human serum samples.
Graphical Abstract Schematic of the electrochemical strategy for adenosine triphosphate detection using aptamer-based target recognition and dendritic DNA nanoassembly amplification
The article describes a colorimetric assay for the determination of thrombin. It is based on the application of a triple enzyme-mimetic activity and a dual aptamer binding strategy. The triple signal amplification relies on oxidation of the chromogenic enzyme substrate 3,3,5,5-tetramethylbenzidine (TMB) that is catalyzed by composites consisting of graphene oxide (GO), gold/platinum nanoparticles (AuPtNP), and aptamer (Apt15), a G-quadruplex/hemin conjugate. The dual-aptamer target binding strategy is based on the fact that thrombin has two active sites to be recognized by its aptamers (Apt15 and Apt29). Magnetic beads (MBs) were modified with Apt29 (Apt29-MB) and then are bound by the GO-AuPtNP-Apt15/G-quadruplex/hemin composites. In the presence of thrombin, Apt29-MB and the GO-AuPtNP-Apt15/G-quadruplex/hemin composites form a sandwich-like superstructure. Thus, the absorbance increases due to the formation of TMB oxide produced by catalysis of the composites. Under optimized conditions, the absorbance at 450 nm increases linearly in the 0.30 to 100 nM thrombin concentration range, and the limit of detection is 0.15 nM. The method is simple, rapid, and does not require complicated instrumentation. Bovine serum albumin, human serum albumin and other proteins were found not to interfere.
Graphical abstract Schematic presentation of the photometric thrombin assay based on a triple enzyme-mimetic activity of combined nanomaterials (consisting of GO, AuPtNPs and the G-quadruplex/hemin DNAzyme) and two aptamers TMB: 3,3,5,5-tetramethylbenzidine, TMBox: 3,3,5,5-tetramethylbenzidine oxide, AuPtNP: gold/platinum nanoparticles).
A study is presented on the binding kinetics and mechanism of the adsorption of dsDNA on citrate-capped gold nanoparticles (AuNPs). Methods include fluorescence titration, isothermal calorimetry (ITC) titration, dynamic light scattering and gel electrophoresis. It is found that the fluorescence of probe DNA (labeled with Rhodamine Green and measured at excitation/emission peaks of 498/531 nm) is quenched by addition of AuNPs. The Stern-Volmer quenching constant (Ksv) is 1.67?×?10^9 L·mol?1 at 308 K and drops with increasing temperature. The quenching mechanism is mainly static. The results of both fluorescence titrations and ITC show negative values for ΔH and ΔS values. This shows ion-induced dipole-dipole interaction to be the main attractive forces between dsDNA and AuNPs, while electrostatic interactions result in repulsion. The repulsive forces lead to a lower affinity between dsDNA and AuNPs (compared to single-strand DNA). It is also found that dsDNA can prevent the aggregation of AuNPs which is accompanied by a color change from red into blue. The visual detection limit with bare eyes for dsDNA1 is 36 pM. Based on these findings, a colorimetric method was developed to detect the proto-oncogene of serine/threonine-protein kinase B-Raf V600E point mutation in HT29, Ec109, A549, Huh-7 and SW480 cell lines.
Graphical abstract Schematic of the salt-induced aggregation of uncapped gold nanoparticles (AuNPs) which leads to a color change from red to blue. If the AuNPs are coated with dsDNA, aggregation is suppressed.
An ultrasensitive conformation-dependent colorimetric assay has been developed for the detection of mercury(II) ions. It is based on the use of exonuclease III (Exo III)-assisted target recycling and gold nanoparticles (AuNPs). In the absence of Hg(II), the hairpin-shaped DNA probe (H-DNA) binds to AuNPs and stabilizes them in solutions of high ionic strength. In the presence of Hg(II), on the other hand, the sticky termini of the H-DNA form a rigid DNA duplex stem with a blunt 3′-terminus. Thus, Exo III is activated as a biocatalyst for selective and stepwise removal of mononucleotides from the 3′-terminus of the H-DNA. As a result, Hg(II) is released from the T?Hg(II)?T complexes. The guanine-rich sequences released from the H-DNA are then self-assembled with potassium ion to form a stable G-quadruplex conformation. In solutions of high ionic strength, this results in aggregation of AuNPs and a color change from red to blue which can be seen with bare eyes. The method is highly sensitive and selective. It has a linear response in the 10 pM to 100 nM Hg(II) concentration range, and the detection limit is as low as 3.2 pM (at an S/N ratio of 3). The relative standard deviation at a level of 0.5 nM of Hg(II) is 4.9% (for n?=?10). The method was applied to the detection of Hg(II) in spiked environment water samples, with recoveries ranging from 92% to 106%.
Graphical abstract A conformation-dependent colorimetric system was fabricated for label-free detection of mercury(II) by utilizing exonuclease III(Exo III)-assisted target recycling and gold nanoparticles (AuNPs).
Patients with prostate cancer and systemic lupus erythematosus exhibit reduced DNase I activity, and patients with myocardial infarction exhibit increased DNase I activity. So the assay of DNase I is of high importance. A colorimetric assay is described here for the determination of the activity of DNase. It is based on strand scission of dsDNA as catalyzed by DNase I. The products of digestion (nucleoside monophosphates) can better stabilize citrate capped AuNPs than dsDNA. In the absence of DNase I, the AuNPs aggregate in presence of NaCl and then display a blue color. In the presence of the analyte (DNase I), AuNPs do not aggregate but rather remain dispersed and display a red color. These findings were exploited to design a DNase I activity assay that is based on the measurement of ratio of absorbances at 520 nm (red) to 650 nm (blue). The detection limit for DNase I activity is found to be 7.1 U?L?1. In our perception, this assay has a large potential with respect to diagnoses of DNase I activity-related diseases and in drug screening.
Graphical Abstract A method is described for the determination of the activity of DNase I. It is based on the capability of nucleoside monophosphates (dNMPs; formed by DNase-catalyzed scission of dsDNA) to stabilize red gold NPs against NaCl-induced aggregation. AuNPs stabilized with dsDNA, in contrast, readily aggregate in presence of NaCl to form blue clusters.
The authors describe a novel assay for the detection of methylated DNA site. Rolling circle amplification and CdSe/ZnS quantum dots with high fluorescence efficiency are applied in this method. The CdSe/ZnS quantum dots act as electron donors, and hemin and oxygen (derived from hydrogen peroxide act as acceptors in photoinduced electron transfer. The assay, best performed at excitation/emission peaks of 450/620 nm, is sensitive and specific. Fluorometric response is linear in the 1 pM to 100 nM DNA concentration range, and the lowest detectable concentration of methylated DNA is 142 fM (S/N =?3). The method is capable of recognizing 0.01% methylated DNA in a mixture of methylated/unmethylated DNA.
Graphical abstract A novel method for methylated sites detection in DNA is established. Rolling circle amplification and photoinduced electron transfer. CdSe/ZnS quantum dots with high fluorescence efficiency act as the electron donor, while G-quadruplex/hemin and hydrogen peroxide derived oxygen act as electron acceptor. It presents a linear response towards 1 pM to 100 nM methylated DNA with a correlation coefficient of 0.9968, and the lowest detectable concentration of methylated DNA was 142 fM, with selectivity significantly superior to other methods.
The authors describe an array for chemiluminescence (CL) based determination of cardiac troponin T (cTnT), an important cardiovascular disease marker. The tracing tag consists of silver nanoparticles (AgNPs) loaded with guanine-rich DNA sequences and detection antibody in a high numerical ratio. The loaded AgNPs were then reacted with hemin to form a hemin/G-quadruplex DNAzyme. A disposable immunosensor array was fabricated by immobilizing capture antibody on corresponding sensing sites on a glass chip. Once a sandwich immunocomplex is formed on the array, the tracing tag catalyzes the CL reaction of the luminol-p-iodophenol and H2O2 system to produce a CL signal, which is collected by a CCD camera. An intuitive CL image is obtained containing all of the spots on the array. Under optimal conditions, the method shows a wide linear range over 4 orders of magnitude (from 0.003 to 270 ng·L?1), a detection limit down to 84 fg·L?1, and a throughput as high as 44 tests·h?1. The results obtained with serum samples are in acceptable agreement with reference values. The AgNP-based tracing tag as well as the immunoassay method shows a promising potential for point-of-care testing for the early clinical diagnosis of cardiovascular disease.
Graphical abstract Schematic presentation of silver nanoparticles (AgNPs) functionalized with hemin/G-quadruplex DNAzyme for highly sensitive chemiluminescence (CL) immunoassay of cardiac troponin T (cTnT) on a glass chip array.
The authors describe an electrochemical DNA nanosensor based on the use of single gold nanowire electrodes (AuNWEs). The probe DNA is immobilized on the AuNWE via Au-S bonds that are formed between thiol-terminated DNA and the gold surface. Single AuNWEs were prepared by an improved laser-assisted pulling method and hydrofluoric acid etching. The nanoelectrodes were characterized by cyclic voltammetry and COMSOL simulation. Square wave voltammetry was used to monitor the DNA hybridization event between probe DNA and target DNA by using Methylene Blue (MB) as an intercalator of dsDNA. Under optimal conditions, the peak current for MB (best measured at a potential of ?0.2 V vs. Ag/AgCl) increases linearly with the logarithm of the analyte concentration in the 1.0 f. to 10 nM range, with a 0.48 fmM detection limit at an S/N ratio of 3. The assay is highly selective, reproducible and stable. Considering the small overall dimensions and high sensitivity, this nanoelectrode potentially can be applied to in-vivo sensing of DNA inside living cells
Graphical abstract Schematic presentation of an electrochemical DNA nanosensor using single gold nanowire electrodes and based on the interaction of thiol-terminated DNA and gold surface. It was used to detect complementary DNA with high selectivity and sensitivity.
The authors have investigated (a) the self-assembly of single-stranded DNA (ssDNA) on glass surfaces, and (b) the interaction of DNA with liquid crystals (LCs) on solid surfaces. The results suggest that ssDNA (compared to dsDNA) on the solid interface causes particularly different orientations in LCs. The LC molecules assume a uniform homeotropic orientation on the surface with a typical surface ssDNA coverage of ~2.4 × 1012 molecules per square cm. Once complementary DNA is hybridized on the surface, the homotropic orientation of the LCs becomes disrupted. This orientation transition can be visually observed by using a crossed polarizer. The findings were exploiting to design an assay for target DNA (= analyte DNA) that has an ~0.1 nM detection limit. The assay is highly selective and can easily differentiate target DNA from single-base mismatch and non-complementary DNA. In our perception, it represents a powerful, label-free and portable DNA detection scheme.
Graphical abstract Schematic illustration of the mechanism for orientation behavior of a liquid crystal film supported on different surfaces. The homeotropic orientation of LC molecules was induced by ssDNA with appropriate surface coverage and was disrupted by ssDNA with lower or higher surface coverage or P1/T1 complex. 5CB: 4-Cyano-4′-pentylbiphenyl. TEA: Triethoxysilylbutyraldehyde.
The authors describe an electrochemical sensing strategy for highly sensitive and specific detection of target (analyte) DNA based on an amplification scheme mediated by a multicomponent nucleic acid enzyme (MNAzyme). MNAzymes were formed by multicomponent complexes which produce amplified “output” signals in response to specific “input” signal. In the presence of target nucleic acid, multiple partial enzymes (partzymes) oligonucleotides are assembled to form active MNAzymes. These can cleave H0 substrate into two pieces, thereby releasing the activated MNAzyme to undergo an additional cycle of amplification. Here, the two pieces contain a biotin-tagged sequence and a byproduct. The biotin-tagged sequences are specifically captured by the detection probes immobilized on the gold electrode. By employing streptavidinylated alkaline phosphatase as an enzyme label, an electrochemical signal is obtained. The electrode, if operated at a working potential of 0.25 V (vs. Ag/AgCl) in solution of pH 7.5, covers the 100 pM to 0.25 μM DNA concentration range, with a 79 pM detection limit. In our perception, the strategy introduced here has a wider potential in that it may be applied to molecular diagnostics and pathogen detection.
Graphical abstract An electrochemical strategy for sequence-specific DNA detection based on multicomponent nucleic acid enzyme (MNAzyme) -mediated signal amplification.
An aptamer based method is described for the electrochemical determination of ampicillin. It is based on the use of DNA aptamer, DNA functionalized gold nanoparticles (DNA-AuNPs), and single-stranded DNA binding protein (ssDNA-BP). When the aptamer hybridizes with the target DNA on the AuNPs, the ssDNA-BP is captured on the electrode surface via its specific interaction with ss-DNA. This results in a decreased electrochemical signal of the redox probe Fe(CN)63? which is measured best at a voltage of 0.188 mV (vs. reference electrode). In the presence of ampicillin, the formation of aptamer-ampicillin conjugate blocks the further immobilization of DNA-AuNPs and ssDNA-BP, and this leads to an increased response. The method has a linear reposne that convers the 1 pM to 5 nM ampicillin concentration range, with a 0.38 pM detection limit (at an S/N ratio of 3). The assay is selective, stable and reproducible. It was applied to the determination of ampicillin in spiked milk samples where it gave recoveries ranging from 95.5 to 105.5%.
Graphical abstract Schematic of a simple and sensitive electrochemical apta-biosensor for ampicillin detection. It is based on the use of gold nanoparticles (AuNPs), DNA aptamer, DNA functionalized AuNPs (DNA-AuNPs), and single-strand DNA binding protein (SSBP).
