Methylene Blue Label Research Articles

A sensitive tobramycin electrochemical aptasensor based on multiple signal amplification cascades

The residue of tobramycin, a broad spectrum antibiotic commonly used in animal husbandry, has evitable impact on human health, which may cause kidney damage, respiratory paralysis, neuromuscular blockade and cross-allergy in humans. Sensitive monitoring of tobramycin in animal-derived food products is therefore of great importance. Herein, a new aptamer electrochemical biosensor for sensing tobramycin with high sensitivity is demonstrated via exonuclease III (Exo III) and metal ion-dependent DNAzyme recycling and hybridization chain reaction (HCR) signal amplification cascades. Tobramycin analyte binds aptamer-containing hairpin probe to switch its conformation to expose the toehold sequence, which triggers Exo III-based catalytic digestion of the secondary hairpin to release many DNAzyme strands. The substrate hairpins immobilized on the Au electrode (AuE) are then cyclically cleaved by the DNAzymes to form ssDNAs, which further initiate HCR formation of lots of long methylene blue (MB)-tagged dsDNA polymers on the AuE. Subsequently electro-oxidation of these MB labels thus exhibit highly enhanced currents for sensing tobramycin within the 5–1000 nM concentration range with an impressive detection limit of 3.51 nM. Furthermore, this strategy has high selectivity for detecting tobramycin in milk and shows promising potential for detect other antibiotics for food safety monitoring.

Introducing common oxazine fluorophores as new redox labels for electrochemical DNA sensors

The electrochemical properties of three oxazine fluorophores, ATTO655, ATTO680 and ATTO700 have been investigated at gold electrodes. They display a reversible or quasi-reversible voltammetric behaviour involving either a 2e−, 2H+ or a 2e−, 1H+ redox process depending on the pH, at a formal potential located in the stability range of thiolate self-assembled monolayers (E°' ≈ −0.33 V vs. Ag|AgCl|3M KCl at pH 7.2). The performance of ATTO655 as redox label for electrochemical nucleic acid sensing was evaluated in a typical E-DNA configuration. The redox label has no detrimental impact on the folding of DNA, as shown with the i-motif forming sequence investigated here. An electron transfer rate constant around 40 s−1 was determined, which is comparable to the values reported for the popular methylene blue label. Hybridisation experiments show a significant signal variation between ssDNA and dsDNA, though it is emphasised that the sign and amplitude of the variation are highly dependent on the electrochemical parameters such as the frequency in square wave voltammetry.

A highly sensitive electrochemical aptasensor for Sonic Hedgehog biomarker based on primer exchange reaction and catalytic hairpin assembly amplifications

Sonic Hedgehog (SHh) protein is commonly over-expressed in various cancers and correlates closely to tumor generation, metastasis and drug resistance, and the detection of SHh has significances for diagnosing various cancers. In this work, by using a new SHh-specific aptamer and combining primer exchange reaction (PER) and catalytic hairpin assembly (CHA), we establish an amplified electrochemical biosensor for sensitive assay of SHh. The combination of SHh and its aptamer results in the release of a strand that can hybridize with the dumbbell-shaped hairpin to trigger PER for generation of lots of new ssDNAs. These strands further initiate CHA cycles to promote confinement of plenty methylene blue (MB)-labeled sequences on the sensor surface. Largely magnified electrochemical current signals from the surface-attached MB labels then enable amplified assay of SHh with a detection limit of 4.1 pM, due to the two-step cyclic amplifications of PER and CHA. Our developed biosensor can also be utilized to assay low concentrations of SHh that is spiked into human serum diluent, verifying its potential for sensitively detecting various molecular markers by replacing the corresponding aptamers for disease diagnosis and prognosis evaluation.

Diazinon electrochemical biosensor mediated by aptamer and nanoscale porous carbon derived from ZIF-8

The development of high-performance sensors for the assay of organophosphate pesticides (OPs) is of significant importance due to their broad usage and harmful effects on mammals. In this study, we designed a simple platform based on aptamer immobilized in an engineered zeolitic imidazolate framework (ZIF) for accurate detection of diazinon (DZN) as a widely used pesticide. To do so, aptamer-functionalized ZIF was constructed by using nanoscale porous carbon ZIF (NPCZIF) as scaffolds to load signal dye, methylene blue (MB), and capped with an aptamer as a bio-gatekeeper via π-π interaction. In this sensor, surface-covering aptamers prevented the release of MB. Therefore, the electrochemical signal of MB in the absence of DZN was low. Upon the addition of DZN, the configuration of the aptamer showed a change, resulting in a large number of MB labels being released from the pores. Therefore, the signal of MB increased. The proposed platform was highly sensitive toward DZN with a detection limit as low as 7.8 fM. The validation of the aptasensor was also studied by analyzing DZN in different food and environmental samples with recovery rates of 95.6–102.6%, indicating the feasibility and reliability of aptasensor.

Ratiometric electrochemical detection of miRNA based on DNA nanomachines and strand displacement reaction.

MicroRNAs (miRNAs) play an important role in regulating gene expression in cells. Abnormal expression of miRNAs has been associated with a variety of diseases. A ratiometric electrochemical method for miRNA detection based on DNA nanomachines and strand displacement reaction was developed. Signal probe with ferrocene label and reference probe with methylene blue label were immobilized on gold nanoparticle (AuNP)-coated magnetic microbeads (AuNP-MMBs). The miRNA triggers the strand displacement reaction and forms a duplex with the protect probe, releasing one end of the DNA walker (DW); the released DW hybridizes with the ferrocene (Fc)-labeled signal probe. The signal probe detached from AuNP-MMBs upon cleavage of the Nb.BbvCI enzyme. The oxidation peak of MB moieties on the reference probe remains unchanged and the signals of Fc moieties on the signal probe are inversely proportional to the concentrations of miRNA. The ratio between Fc moieties at 0.35 V and MB moieties at -0.22 V (vs. Ag/AgCl) was used to quantify the expression level of miRNA with a detection limit down to 0.12 fM. The ratiometric assay possesses a strong ability to eliminate interference from environmental changes, thus offering the high selectivity of miRNA from the complexed biosystems, holding great significance for miRNA sensing. A ratiometric assay with high selectivity of miRNA has been developed based on DNA nanomachines and strand displacement reaction.

Target-driven assembly of DNAzyme probes for simultaneous electrochemical detection of multiplex microRNAs.

In this work, we employed target-driven assembly of a Mg2+-dependent DNAzyme to develop an ultrasensitive electrochemical biosensor for the simultaneous detection of miRNA-21 and miRNA-141. The target miRNAs could hybridize with two partial DNAzymes, facilitating the formation of a stable and active Mg2+-dependent DNAzyme. With the help of the Mg2+ cofactor, the DNAzyme could circularly cleave the ferrocene (Fc) or methylene blue (MB) labelled hairpin probes and release Fc and MB labels from the electrode surface, which could significantly amplify the current suppression to achieve multiple detection of small amounts of miRNA-21 and miRNA-141. This electrochemical biosensor showed high sensitivity and selectivity for the simultaneous detection of miRNA-21 and miRNA-141. Furthermore, the proposed method was also successfully applied for the determination of miRNA-21 and miRNA-141 from diluted serum samples. Overall, the proposed sensor showed several considerable advantages including simple preparation, high sensitivity, and enzyme-free signal amplification. Therefore, the proposed electrochemical biosensor could be used as a highly efficient amplification strategy for simultaneous detection of various miRNA biomarkers in bioanalysis and clinical diagnostics.

DNA Nanosieve-Based Regenerative Electrochemical Biosensor Utilizing Nucleic Acid Flexibility for Accurate Allele Typing in Clinical Samples.

Herein, an interface-based DNA nanosieve that has the ability to differentiate ssDNA from dsDNA has been demonstrated for the first time. The DNA nanosieve could be readily built through thiol-DNA's self-assembly on the gold electrode surface, and its cavity size was tunable by varying the concentration of thiol-DNAs. Electrochemical chronocoulometry using [Ru(NH3)6]3+ as redox revealed that the average probe-to-probe separation in the 1 μM thiol-DNA-modified gold electrode was 10.6 ± 0.3 nm so that the rigid dsDNA with a length of ∼17 nm could not permeate the nanosieve, whereas the randomly coiled ssDNA could enter it due to its high flexibility, which has been demonstrated by square wave voltammetry and methylene blue labels through an upside-down hybridization format. After combining the transiently binding characteristic of a short DNA duplex and introducing a regenerative probe (the counterpart of ssDNA), a highly reproducible nanosieve-based E-DNA model was obtained with a relative standard deviation (RSD) as low as 2.7% over seven cycles. Finally, we built a regenerative nanosieve-based E-DNA sensor using a ligation cycle reaction as an ssDNA amplification strategy and realized one-sensor-based continuous measurement to multiple clinical samples with excellent allele-typing performance. This work holds great potential in low-cost and high-throughput analysis between biosensors and biochips and also opens up a new avenue in nucleic acid flexibility-based DNA materials for future applications in DNA origami and molecular logic gates.

Electrode immobilization-free and sensitive electrochemical sensing of thrombin via magnetic nanoparticle-decorated DNA polymers

Abstract The development of sensitive and simple sensing strategies for trace proteins has received increasing attentions in the diagnosis and treatment of diseases. We describe here an electrode immobilization-free and homogeneous electrochemical method for sensitively detecting thrombin in human serums. The strategy relies on target-promoted proximity binding hybridization chain reaction (HCR) formation of magnetic nanoparticle (MNP)-decorated DNA polymers. Simultaneous binding of thrombin by two distinct aptamers increases their local concentrations and enables the proximity binding-induced strand displacement reaction, which leads to subsequent initiation of HCR between the methylene blue (MB)- and biotin-labeled hairpins into long DNA polymers. The streptavidin-modified MNPs further bind the biotin moieties to form the MNPs/DNA polymers. Subsequent accumulation of the MNPs/DNA polymers on the AuNP-deposited electrode can thus yield substantially enhanced current, due to the oxidation of the many MB labels, for highly sensitive detection of thrombin ranging from 5 pM∼50 nM with a detection limit of 1.1 pM in a simple electrode immobilization-free way. Selective interrogation of low levels of thrombin in diluted human serums was also verified, revealing its potential for convenient and ultrasensitive monitoring of a variety of protein biomarkers for disease diagnosis at the early stages.

A reagentless electrochemical sensor for aflatoxin B1 with sensitive signal-on responses using aptamer with methylene blue label at specific internal thymine

Aptamer electrochemical sensors using immobilized aptamers with redox tag rely on the target binding-induced changes of current signal on electrode, offering advantages in operation convenience, no separation, rapidity, and sensitivity. Usually, the redox tag is placed on aptamer terminal, however, sometimes the terminal label may be insensitive to target-binding and fail to generate sensitive responses. The redox tag methylene blue (MB) labeled on different sites of aptamer may experience distinct changes in local environment, distance to electrode, or interactions with aptamer bases during affinity binding, which affect the current signal. Thus, it is possible to construct aptamer electrochemical sensors with sensitive and significant responses to targets by screening a series of sites (e.g., internal thymine T) of the aptamer and placing MB tag on a specific site of the aptamer. With this strategy, we successfully fabricated an electrochemical sensor on gold electrode for rapid, reagentless, and sensitive detection of aflatoxin B1 (AFB1), an important mycotoxin causing great health risks, by using a 26-mer DNA aptamer with MB on an internal T site (e.g., 18th T) and a thiol moiety at 5′ terminal. This sensor generated remarkable signal-on responses to AFB1, allowed a detection limit of 6 pM, and enabled detection of AFB1 in wine, milk and corn flour samples. This sensor can be well regenerated by rinsing with deionized water and reused, and shows good stability. This sensor and the demonstrated strategy are promising in wide applications.

Engineering uranyl-chelating peptides from NikR for electrochemical peptide-based sensing applications

We have, for the first time, designed three uranyl-chelating peptides by modeling after the uranyl binding pocket found in a mutated Ni(II)-dependent transcriptional repressor (NikR). All three thiolated and methylene blue (MB)-modified peptides, NikR-5, NikR-11, and NikR-15, contain the five core amino acids responsible for target recognition, but the two longer peptides have either one or two additional glycine residues in between the five amino acids. These three peptide probes were then used in the fabrication of electrochemical peptide-based (E-PB) uranyl ion (U(VI)) sensors, with the goal of elucidating the effects of the added glycine residues and probe flexibility on target recognition. The sensing mechanism is similar to other “signal-off” E-PB sensors, in which binding of the target rigidifies the probe, resulting in a decrease in the redox signal from the tethered MB label. Although all three sensors responded to U(VI), the NikR-15 sensor's behavior was irreproducible and thus precluded from the rest of the study. The NikR-11 sensor showed the largest response to U(VI), whereas the NikR-5 sensor showed higher specificity for U(VI). The limit of detection was 50 nM for both sensors, which is well below the U.S. Environmental Protection Agency maximum contaminant level for uranium. Both sensors were further tested and proven functional in a 50% synthetic aquifer sample. Overall, the NikR-11 sensor, fabricated with the 11-amino acid probe, is deemed the optimal design. Incorporating glycine residues is a strategic way to lengthen the peptide slightly to improve target binding. This simple yet versatile approach to recreating binding pockets on electrode surfaces can potentially be employed in the design of other E-PB and surface-based metal ion sensors.

Peptide nucleic acid-based electrochemical biosensor for simultaneous detection of multiple microRNAs from cancer cells with catalytic hairpin assembly amplification

Herein we demonstrated a facile electrochemical method for simultaneous detection of miRNA21 and miRNA155 using a peptide nucleic acids (PNAs)-modified gold electrode coupled with the target-catalyzed hairpin assembly (CHA) strategy. In the presence of the target miRNA, the CHA was triggered selectively between two hairpins with one ferrocene (Fc) or methylene blue (MB) labelled. The resulting redox-active group modified CHA products (Fc-CHA21 or MB-CHA155) were then specifically captured by the PNA probes (PNA21 or PNA155) attached on the surface of a gold electrode, which bring the Fc and MB labels into close proximity to generate apparently enhanced electrochemical signals for sensitive and simultaneous detecting of low amount miRNA21 and miRNA155 in cancer cells. This assay was highly selective for discriminating miRNAs with similar sequences and has detection limits of 2.49 fM and 11.63 fM for miRNA21 and miRNA155, respectively. The feasibility of the method for sensitive determination of miRNA21 and miRNA155 from human cancer cells was also demonstrated. This method thus has great potential to be applied for simultaneous detecting of a variety of miRNA biomarkers for clinic applications due to its simple, sensitive and accurate features.

Two kanamycin electrochemical aptamer-based sensors using different signal transduction mechanisms: A comparison of electrochemical behavior and sensing performance

Two typical kanamycin-A (KAN-A) electrochemical aptamer-based sensors employing different signal transduction mechanisms were deliberately designed and constructed with a similar structure. One sensor (sensor-1) was based on the classical probe conformation changing mode (PCCM) with a methylene blue (MB) label used as an electrochemical tag; the other sensor (sensor-2) used the target-induced signal probe shifting (TISPS) method with a free MB label in the assay solution. The difference in signal transduction mechanisms resulted in big differences in basic electrochemical behavior and comprehensive sensing performance. The results show that both sensor types exhibit different electrochemical behavior in square wave voltammetry, cyclic voltammetry, and in sensitivity, with detection limits of 3.0 nM for sensor-1 and 60.0 pM for sensor-2 in buffer. When validated for practical and quantitative detection of tap water and milk samples, both sensing methods performed well with detection limits of <260 nM and measurement times of <40 min. In addition, accuracy was good with mean recoveries of 72.3–92.6% and precision was acceptable with a relative standard deviation (RSD) of ≤14.2%. The basis for the similarities and differences in performance is also presented.

Effects of redox label location on the performance of an electrochemical aptamer-based tumor necrosis factor-alpha sensor

We report the development of an electrochemical aptamer-based sensor for real time detection of tumor necrosis factor-alpha. The focus of this study is to evaluate the effects of the redox label location on the overall sensor performance, including sensor stability, detection limit, reusability, and selectivity. Three aptamer probes, each labeled with methylene blue (MB) at a specific location, were designed and employed in the fabrication of the sensors. Among the three sensors, the sensor fabricated using an aptamer with the MB label located at the distal end has a detection limit of 100 pM and is regenerable. The sensor fabricated using an aptamer with an internal MB modification has a detection limit of 10 nM and is not regenerable. Both sensors can be employed in complex biological samples such as 50% urine and 50% saliva. However, the sensor fabricated with an aptamer with the MB label located at the proximal end suffers from poor reproducibility and is highly unstable, thus limiting its application as a sensor. On the bases of these results, placing the MB label at the distal end of the aptamer probe appears to be the most advantageous for this sensor design for it does not interfere with monolayer formation and target binding.

A signal-on electrochemical aptasensor for highly sensitive and specific detection of kanamycin based on target-induced signaling probe shifting mechanism

An electrochemical displacement-based aptasensor was developed for the detection of kanamycin-a (KMY-A). Based on the target-induced signaling probe shifting mechanism, this aptasensor shows a “signal on” change with a simple 3-probe structure. One no-thiolated capture probe and one thiolated assistant probe are attached onto the Au electrode surface in a short duplex state, and one methylene blue (MB)-labelled signaling probe is free in the detection solution. Before adding the KMY-A, a very small background signal is obtained with low efficient electron transfer because of the MB labels on the signaling probe far from the electrode surface. After adding the KMY-A, the more stable capture probe/KMY-A complex formed between the capture probe and the KMY-A undoes the former short duplex to release the assistant probe, which induces the formation of another assistant probe/signaling probe duplex. This structure change can shift the MB labels close to the electrode surface with much higher efficient electron transfer, thereby leading to a big detection signal. Under the optimized fabrication and detection conditions, the sensor features a high sensitivity for KMY-A with the detection limit of 3.3 pM and a fast sensing speed with the response time of ∼9 min. In addition, it is worth mentioning that the target discrimination ability of sensor can be improved through using longer assistant probe to strengthen the hybridization force between the capture probe and the assistant probe, which is helpful for establishing the special detection system with high specificity requirement.

The Impact of the Position of the Redox Label on Charge Transfer and Hybridization Efficiency at DNA Interfaces

AbstractA common electrode construct for electrochemical DNA biosensors consists of tethering a redox‐label‐modified single stranded DNA to the electrode and comparing the electrochemical response of a redox probe before and after hybridization. Despite the analytical advantages of this construct, contradictory results are found in the literature regarding to the effect of redox‐labeling position on the charge transfer process happening at a DNA interface. Herein a contribution to this discussion is made by investigating how a methylene blue redox label placed at different positions along a probe strand of DNA affects the DNA hybridization efficiency. For that, a second redox label, a ferrocene molecule, was attached to the target DNA at a fixed position such that upon hybridization it is positioned at the distal end of the DNA monolayer. The dual label strategy allowed us to explore, for each methylene blue labeling position, how the binding‐induced diminution in the methylene blue signaling is associated with the appearance of the ferrocene signaling after hybridization. It was found that the location of the methylene blue redox label in the DNA interface can affect the hybridization efficiency.

Hexavalent Chromium as an Electrocatalyst in DNA Sensing.

We report for the first time the use of hexavalent chromium (Cr(VI)) as an electrocatalyst in electrochemical DNA sensing. For both stem-loop probe and linear probe electrochemical DNA sensors, the increase in probe rigidity upon target hybridization alters the accessibility of Cr(VI) to the methylene blue label on the surface-immobilized DNA probes. This change results in an enhancement in the electrocatalytic current when the sensors are interrogated using cyclic voltammetry at a slow scan rate. The incorporation of this electrocatalyst does not affect the normal "signal-off" sensing behavior observed in alternating current voltammetry; instead it enables simultaneous "signal-on" and "signal-off" detection of the target, while maintaining noted attributes of this class of folding- and dynamics-based sensors such as reusability and high selectivity. It is also capable of improving the limit of detection of the sensors by an order of magnitude. Importantly, this accessibility-based electrocatalytic sensing strategy is versatile and can potentially be used with other folding- and dynamics-based electrochemical biosensors.

A reagentless and reusable electrochemical aptamer-based sensor for rapid detection of Cd(II)

We report the design and fabrication of a “signal-on” electrochemical aptamer-based sensor for detection of cadmium (Cd(II)). A 36-base thiolated and methylene blue (MB)-modified aptamer was used in the fabrication of the sensor. In the absence of Cd(II), the surface-immobilized aptamer probe is presumed to be partially folded. Upon binding to Cd(II), the aptamer probe changes its confirmation and flexibility, which alters the electron transfer kinetics between the tethered MB label and the underlying electrode, resulting in an increase in the MB signal. Parameters affecting the detection of Cd(II) were systematically optimized and the electrochemical performance of the sensor was assessed using both alternating current voltammetry and cyclic voltammetry. The sensor has demonstrated good selectivity and can be employed directly in complex samples such as 50% saliva. Furthermore, it has a fast-response time; the analysis can be completed in ~1min. Similar to other folding- and dynamics-based electrochemical biosensors, it is also regenerable and reusable. With further optimization, it could find applications in the analysis of Cd(II) in biological and environmental samples.

A dual-probe surface-co-immobilization-based electrochemical sensor for highly sensitive and tunable specific detection of a designated DNA

A “signal on” electrochemical sensing method was established for the detection of a designated 24-base DNA. This sensor system shows a simple structure with only composing of three probes. One capturing probe (CP) and one assistant probe (AP) are co-immobilized onto the Au electrode surface in a short CP/AP duplex state, and one methylene blue (MB) modified signaling probe (SP) is free in the detection solution. Before adding the target, a low background signal helpful for improving the sensitivity is obtained with low efficient electron transfer because of the MB labels far from the electrode surface; after adding the target, the longer and more stable CP/target duplex formed between CP and the target undoes the former CP/AP duplex to release the AP, which induces the formation of another AP/SP duplex. This structure can drive the MB labels close to the electrode surface with much higher efficient electron transfer, thereby producing a large detection signal. The proposed sensor features a high sensitivity with the detection limit (DL) of ∼10 fM for the target. When used for discriminating the mismatched target, the sensor also exhibits a good ability and its high adjustability.

DNA-mediated strand displacement facilitates sensitive electronic detection of antibodies in human serums

We describe here the development of a sensitive and convenient electronic sensor for the detection of antibodies in human serums. The sensor is constructed by self-assembly formation of a mixed monolayer containing the small molecule epitope conjugated double stranded DNA probes on gold electrode. The target antibody binds the epitope on the dsDNA probe and lowers the melting temperature of the duplex, which facilitates the displacement of the antibody-linked strand of the duplex probe by an invading methylene blue-tagged single stranded DNA (MB-ssDNA) through the strand displacement reaction and leads to the capture of many MB-ssDNA on the sensor surface. Subsequent electrochemical oxidation of the methylene blue labels results in amplified current response for sensitive monitoring of the antibodies. The antibody assay conditions are optimized and the sensor exhibits a linear range between 1.0 and 25.0nM with a detection limit of 0.67nM for the target antibody. The sensor is also selective and can be employed to detect the target antibodies in human serum samples. With the advantages of using small molecule epitope as the antibody recognition element over traditional antigen, the versatile manipulability of the DNA probes and the unique properties of the electrochemical transduction technique, the developed sensor thus hold great potential for simple and sensitive detection of different antibodies and other proteins in real samples.

Highly Sensitive Voltammetric Thrombin Aptamer Sensor Based on the Synergistic Effect of Doping/Depositing Gold Nanoparticles in Polydopamine Film

AbstractA highly sensitive square‐wave voltammetric thrombin (TB) aptamer sensor was developed using functional polydopamine (PD) film by doping and depositing gold nanoparticles into the bulk and the surface of PD. The aptamer sensor was fabricated by immobilizing a thiolated TB‐binding aptamer (TBA) on the AuNPs‐doped/deposited PD film. AuNPs‐supported methylene blue labels were used for the detection of human α‐TB. Under the optimized conditions, the aptamer sensor’s dynamic range and the detection limit were determined to be 2.0 pM–50 nM and 0.97±0.06 pM, respectively. Finally, the proposed aptamer sensor was successfully examined in human serum samples and satisfactory results were obtained.