G-quadruplex DNAzyme Research Articles

A DNAzyme-catalyzed label-free aptasensor based on multifunctional dendrimer-like DNA assembly for sensitive detection of carcinoembryonic antigen

Carcinoembryonic antigen (CEA) is an important malign tumor marker. In this study, a simple, label-free and antibody-free aptasensor was fabricated based on a multifunctional dendrimer-like DNA nanoassembly. The DNA nanoassembly was embedded with multiple G-quadruplex DNAzyme motifs and a hanging CEA aptamer motif. It was prepared from short DNA sequences by autonomous-assembly. The aptasensor was prepared simply by self-assembly of a capture DNA (cpDNA) on a gold electrode, followed by hybridization with a CEA aptamer (AptGAC-P). CEA as a model target was detected through competitive binding of CEA with AptGAC-P, exposing cpDNA to bind with the DNA nanoassembly. The detection process only contains 2 incubation steps. The high load of G-quadruplex DNAzyme motifs and their catalytic activity resulted in an amplified and label-free differential pulse voltammetry (DPV) electrochemical signal. The peak current correlated linearly with the CEA concentration, with a linear range of 2–45 ng mL−1, and an LOD value of 0.24 ng mL−1. The aptasensor showed high specificity and reproducibility, and retained 96.5% of detection signal intensities after 31 days of storage. The recovery rates for spiked CEA in human serum were within 100 ± 5%, and the coincidence rates for clinical human serum samples with ELISA kits were 80.7–111%. Conceivably, possessing simplicity, sensitivity, reproducibility, storage stability, and accuracy, the aptasensor should be a very prominent and applicable tool for clinical CEA detection and cancer diagnosis, and is promisingly applicable as a platform for detecting other targets of interests.

A visual cardiovascular biomarker detection strategy based on distance as readout by the coffee-ring effect on microfluidic paper

We first reported a novel visualized instrument-free signal conversion method on paper for cardiovascular biomarker detection based on the coffee-ring effect (CRE). Our strategy was based on the principle that cTnI was captured by DNA probe decorated detection antibody to form sandwich complex, which could hybridize with H1 probes to form G-quadruplex DNAzyme. As a peroxidase mimic, G-quadruplex DNAzyme could catalyze colorimetric reaction and generate colored products, which were converted to the end edge of the paper by evaporation-driven flow, forming a visible colored band. Hence, the resulted colored signals were converted to distance signal by CRE, so that the quantification of cTnI could be measured easily only by a ruler. Our results demonstrated that the length of the colored bands was proportional to the concentration of cTnI, with a limit of detection of 1.0 pg/mL. Furthermore, this strategy not only showed advanced performance to discriminate cTnI from complex samples but also exhibited high accuracy with standard method in real sample. Hence, these results demonstrated that this method established a promising equipment-free and visualized platform for detecting various kinds of genetic diseases and biomarkers, further promoting the development of point-of-care testing (POCT) in biomedicine.

Ultrasensitive pathogenic bacteria detection by a smartphone-read G-quadruplex-based CRISPR-Cas12a bioassay

Foodborne diseases, caused by pathogenic bacteria, severely threaten global human health and cause a financial burden. Rapid, sensitive and on-site detection of pathogenic bacteria is significant. The existing methods have different defects, such as time-consuming and inconvenient. In this study, we developed a G-quadruplex-based CRISPR-Cas12a bioassay for pathogenic bacteria detection with high sensitivity and visualization capability. Salmonella was used as the detection model. Simply, the amplicons of Salmonella specific invA gene activated the trans-cleavage activity of Cas12a and triggered CRISPR-Cas12a based indiscriminate degradation of single-stranded DNAs (ssDNAs). The ssDNAs were designed with the guanine-rich sequence and formed a stable G-quadruplex DNAzyme by adding K+. This DNAzyme could catalyze the TMB-H2O2 reaction in the presence of hemin, leading to an increase in absorbance at 454 nm and a color change. This change can be readily differentiated by the naked eyes as well as a smartphone with a Color Picker App. With this strategy, the limit of detection (LOD) for Salmonella was 1 CFU/mL with no cross-reactivity. A linear relationship (R2 = 0.993) between the absorbance and the concentration of Salmonella was obtained. Furthermore, G-quadruplex-based CRISPR-Cas12a bioassay was successfully applied for sensing Salmonella in real food samples. This work not only expands the reach of CRISPR-Cas based biosensing but also provides a novel pathogenic bacteria detection method with high sensitivity, specificity and on-site capability.

G-quadruplex DNAzyme as peroxidase mimetic in a colorimetric biosensor for ultrasensitive and selective detection of trace tetracyclines in foods

The colorimetric method can determine the initial results even by the naked eyes, but its main challenge for antibiotics detection in food at present is the relatively low sensitivity. Herein, an ultrasensitive colorimetric biosensor based on G-quadruplex DNAzyme was firstly proposed for the rapid detection of trace tetracycline antibiotics like tetracycline, oxytetracycline, chlortetracycline and doxycycline. DNAzyme composed of hemin and G-quadruplex has peroxidase-like activity, and tetracyclines can combine with hemin to form a stable complex and reduce catalytic activity, making the color of solution changes from yellow to green. The limits of detection (LOD) of the proposed colorimetric biosensor for tetracyclines is determined as low as 3.1 nM, which is lower than most of the other colorimetric methods for antibiotics detection. Moreover, the average recovery range of tetracyclines in actual samples is from 89% to 99%, indicating that such strategy may has bright application prospects for tetracyclines detection in foods.

An electrochemical dual-aptamer biosensor based on metal-organic frameworks MIL-53 decorated with Au@Pt nanoparticles and enzymes for detection of COVID-19 nucleocapsid protein

The development of COVID-19 detection strategies with high sensitivity and selectivity are urgent for early diagnosis. Herein, we constructed an electrochemical dual-aptamer biosensor based on the metal-organic frameworks MIL-53(Al) decorated with Au@Pt nanoparticles and enzymes to determine SARS-CoV-2 nucleocapsid protein (2019-nCoV-NP) via co-catalysis of the nanomaterials, horseradish peroxidase (HRP) and G-quadruplex DNAzyme. First, the two thiol-modified aptamers (N48 and N61), as recognition elements, were immobilized on the surface of gold electrode (GE) to capture the biomarker 2019-nCoV-NP. Then, the nanomaterial composites Au@Pt/MIL-53 (Al) were decorated by HRP and hemin/G-quadruplex DNAzyme as signal nanoprobe. The designed nanoprobe was applied to amplifying the aptasensor signal via co-catalyzing the oxidation of hydroquinone in the presence of hydrogen peroxide. Finally, the aptamer-protein-nanoprobe sandwich electrochemical detection system was fabricated on the GE surface. The results demonstrated that this aptasensor had a wide linear range from 0.025 to 50 ng mL−1 and the detection limit was 8.33 pg mL−1 for 2019-nCoV-NP. This aptasensor has great potential in the early diagnosis of COVID-19 with high sensitivity, selectivity and reliability.

Highly Selective Detection of K+ Based on a Dimerized G-Quadruplex DNAzyme.

Potassium ion (K+) plays a crucial role in biological systems, such as maintaining cellular processes and causing diseases. However, specifically, the detection of K+ is extremely challenging because of the coexistence of the chemically similar ion of Na+ under physiological conditions. In this work, a K+ specific biosensor is constructed on the basis of a dimerized G-quadruplex (GQ) DNA, which is promoted by K+, and the enzymatic activity of the resulting DNAzyme depends on the concentration of the K+. The K+ in a 1-200 mM concentration range can be selectively detected by visual color, UV-Vis absorbance or fluorescence even if the concentration of the accompanying Na+ is up to 140 mM at an ambient condition up to 45 °C. In addition, this system can also be used to selectively detect NH4+ in a 5-200 mM concentration range. This dimerized DNAzyme offers a new type of biosensor with a potential application in the biological system.

A novel assay strategy based on isothermal amplification and cascade signal amplified electrochemical DNA sensor for sensitive detection of Helicobacter pylori

A novel and sensitive Helicobacter pylori assay strategy based on linear isothermal amplification reaction (LIAR) and DNAzymes catalyzed DNA sensor was developed. A 17 nt target DNA (tDNA) specific in the H. pylori genome was selected and used as primer in the LIAR. A template DNA was designed to produce an amplicon with Mg2+-dependent and RNA-cleaving DNAzyme (Md-Dz) activity. The amplicon (i.e. Md-Dz) was then detected with the DNA sensor. The DNA sensor was fabricated simply through immobilization of a hairpin DNA that contains both Md-Dz substrate and G-quadruplex DNAzyme (Gq-Dz), on gold electrodes. The Gq-Dz possesses peroxidase activity, catalyzing the electro-redox of the electrochemical signal probe hydroquinon. The LIAR, Md-Dz catalytic reaction, and DNA sensor fabrication and detection processes were characterized and optimized. The LIAR amplification efficiency is as high as ~5 × 105 fold, and the assay strategy is very sensitive, selective and repetitive. The differential pulse voltammetry (DPV) detection signals linearly correlate to the logarithmic tDNA concentration in the range of 0.1–10,000 fM, with a limit of detection (LOD) of 34 aM. The detection linear range for H. pylori genomic DNA is 2.1–67.2 pg, with an LOD of 1.3 pg. Muted DNA sequences and other pathogenic bacteria did not result in interferences. In addition, the DNA sensor is very stable in storage. More importantly, the DNA sensor and the assay strategy could be universally applicable to detect any biological sample and short DNA sequence of interests.

Temperature-robust and ratiometric G-quadruplex proximate DNAzyme assay for robustly monitoring of uranium pollution and its microbial biosorbents screening

Uranium pollution in environment and food chain is a serious threat to public security and human health. Herein, we proposed a temperature-robust, ratiometric, and label-free bioassay based on G-quadruplex proximate DNAzyme (G4DNAzyme), accommodating us to precisely monitor uranium pollution and biosorption. The proximity of split G-quadruplex probes was proposed to sense UO22+-activated DNAzyme activity, thus eliminating the use of chemically labeled nucleic acid probes. And the simultaneous monitoring of G-quadruplex and double-stranded structures of DNAzyme probes contributed to a ratiometric and robust detection of UO22+. Particularly, the separation of enzymatic digestion and fluorescence monitoring endued a robust and highly responsive detection of UO22+ upon the temperature of enzymatic digestion process ranged from 18° to 41 °C. Consequently, G4DNAzyme assay allowed a robust, label-free and ratiometric quantification of uranium. We demonstrated the feasibility of G4DNAzyme assay for estimating uranium pollution in water and aquatic product samples. Ultimately, G4DNAzyme assay was adopted to serve as the platform to screen bacterial species and conditions for uranium biosorption, promising its roles in uranium associated biosafety control.

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

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

DsDNA/ssDNA-switchable isothermal colorimetric biosensor based on a universal primer and λ exonuclease

Herein, a dsDNA/ssDNA-switchable isothermal colorimetric biosensor was established. In our design, a versatile linker domain invades into recombinase polymerase amplification (RPA) amplicons via a reverse primer with an additional linker. To activate effective amplification, a universal primer (U-P) is introduced to address the impaired efficiency in the invasion process. After billions of dsDNA amplicons with the universal linker accumulate, λ exonuclease acts as a converter and mediates the transformation from dsDNA to ssDNA for launching efficient DNA self-assembly without signal leak. The newly formed G-rich sequences in the ladder-like assembly fold into G-quadruplex DNAzyme for visual signal. A model pathogen (Salmonella spp.) and a model plant (MON810 maize) were detected by our sensor with excellent sensitivity and selectivity, demonstrating the universality. The linearity of Salmonella spp. was y (A450) = 0.1571x (the logarithm of concentrations) + 0.4452 (R2 = 0.9903) from 101 cfu/mL to108 cfu/mL with the detection limit of 3 cfu/mL; and the visible semi-quantified detection limit of MON810 maize was 0.1 %. Meanwhile, a recovery and a selective experiment were performed in yogurt starter with Salmonella spp.. Generally, this study provides a dsDNA/ssDNA-switchable colorimetric strategy based on a universal primer and λ exonuclease. A project of compositive suitcase, constituted by a DNA crude extract kit, an isothermal colorimetric kit and a portable spectrophotometer, was developed for versatile detection of different targets for a mobile laboratory. This shows great promise for on-site bioassays in food safety, environmental monitoring and clinical diagnosis.

A novel aptasensor based on HCR and G-quadruplex DNAzyme for fluorescence detection of Carcinoembryonic Antigen

In this paper, a rationally designed aptasensing platform based on Hybridization Chain Reaction (HCR) and G-quadruplex DNAzyme for the fluorescence detection of Carcinoembryonic Antigen (CEA) has been developed. In the presence of target CEA, the aptamer sequence in Aptamer Probe (AP) specifically bound to CEA, resulting in the AP conformation change and thus releasing initiator, which triggered the autonomous cross-opening of Hairpin 1 (H1) and Hairpin 2 (H2) that yielded extended nicked double-stranded DNA via HCR. Upon the addition of hemin, G-rich segments at the end of H1 and H2 self-assembled into the peroxidase-mimicking hemin/G-quadruplex DNAzymes, which catalyzed the hydrogen peroxide-mediated oxidation of thiamine to achieve fluorescence detection of CEA. The HCR product, and the formation and catalytic performance of DNAzyme were characterized by agarose gel electrophoresis, UV–vis spectroscopy and fluorescence spectroscopy, respectively. Under optimal conditions, the fluorescent aptasensor showed a linear relationship ranging from 0.25 to 1.5 nM toward CEA with a detection limit of 0.2 nM. In addition, this aptasensor exhibited high selectivity for CEA without being affected by other interfering proteins, such as IgG, AFP and PSA. Furthermore, this proposed aptasensor was successfully applied to CEA analysis in diluted human serum samples. It is believed that this strategy has a promising potential in biochemical analysis and clinic application.

Use of Tris-NH4Cl in modified G4/hemin DNAzyme assay with duplexed probes extends life of colorized radical product

The split G-quadruplex/hemin DNAzyme exhibits peroxidase-like activity and catalyzes the oxidation of colorless 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS2−) to the green-colored radical ABTS∙− in the presence of H2O2. The two major challenges to overcome are the relatively low catalytic activity and the short color retention after catalytic action with the substrate ABTS2−. We improved the sensitivity of the G-quadruplex DNAzyme-based DNA assay by utilizing split (duplex) DNAzyme hybridization and adjusting reagent concentrations under limited temperature and pH conditions. Significant improvements in sensitivity and stability were achieved using the activator Triton X-100 and NH4+ as compared to traditional K+. In addition, we demonstrate that the rapid color bleaching of the catalytic substrate ABTS2− is due to the disproportionation of the green-colored reaction product ABTS∙− by H2O2 rather than the degradation of hemin by H2O2. Since residual H2O2 remains after the primary catalytic reaction, the Tris-NH4Cl buffer preserves the radical by removing H2O2 that remains after completion of the primary reaction before it scavenges the radical product. This improved split G-quadruplex DNAzyme formulation can be used to detect various targets conveniently and rapidly, and the test results are stable for more than three days.

Highly sensitive remote biosensor for the determination of lead (II) ions by using nanoporous anodic alumina modified with DNAzyme

Herein, we have designed a biosensor based on interferometric reflectance spectroscopy (IRS) for the lead (II) ion (Pb2+) ions sensing. For this purpose, the electrochemically fabricated nanoporous anodic alumina (NAA) was fabricated and then functionalized with amino-containing silane (NAA-NH2). After that, the amino-terminal G-rich DNA single strands were immobilized into the pores of NAA-NH2 by using glutaraldehyde (Glu). Finally, the NAA modified with G-rich DNA single strands was immersed in a solution containing hemin and potassium ions, fabricating a DNAzyme sensor (NAA-NH-Glu-NH-DNAzyme). The proposed biosensor oxidized 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) solution to green color ABTS˙− anion radical solution in the presence of hydrogen peroxide. The generated green color ABTS˙− anion radical solution absorbed the white light and therefore the intensity of the reflected light from NAA to the CCD decreased. However, in the presence of Pb2+ ions, the catalytic property of the proposed biosensor decreased. In the presence of Pb2+ ions, the concentration of the generated green color ABTS˙− anion radical solution decreased because of releasing the hemin cofactor during the transformation of the K+-stabilized hemin- G-quadruplex DNAzyme into the Pb2+-stabilized G-quadruplex. Therefore, the intensity of the reflected light to the detector increased. This increase in the intensity of the reflected light to the detector consequent on the increase in the peak area of the IRS. The biosensor exhibited a good response to Pb2+ ions with a linear range from 50 to 3200 nM with a low detection limit of 12 nM (2.6 ppb).

A visual Hg2+ detection strategy based on distance as readout by G-quadruplex DNAzyme on microfluidic paper.

In this work, we have developed a simple, fast and visual Hg2+ detection strategy based on distance as readout on paper chip by the Hg2+-mediated formation of G-quadruplex-hemin DNAzymes. In the presence of Hg2+, the two oligonucleotides hybridize to form G-quadruplex DNA by T-Hg2+-T base pair, which was able to bind hemin to form the catalytically active G-quadruplex-hemin DNAzymes. Once DNAzymes were added to react with the precipitated 3,3,5,5-tetramethyl benzidine (TMB) immobilized on the sample area, a visible color band was produced, and the formed length was positively correlated with the concentration of Hg2+. This biosensor is capable of selectively detecting mercuric ions with good reproducibility and satisfactory dynamic range. The limit of detection was low to 0.23nM. Therefore, this strategy not only provides a visual and quick screen of Hg2+, but also shows a promising future in monitoring analysis of other metal ions in POC diagnostic field.

Entropy-driven spliced DNA walking machine for label-free electrochemical detection of antibiotics

Herein, we have integrated an entropy-driven DNA walking machine into electrochemical platform for label-free sensing of antibiotics. As far as we know, this work is the first time that entropy-driven DNA machine has been used for antibiotics detection. The designed DNA machine is activated by a target-triggered catalytic hairpin assembly to form a spliced DNA walker long-arm. Then the autonomous and progressive spliced walk long-arm on electrode surface via the toehold-mediated strand displacement reaction induces continuous release of signal probe (SP) from three-stranded DNA complex. The freedom of SP leads to the generation of abundant G-quadruplex DNAzyme which can catalyze decomposition of hydrogen peroxide, so an extremely intense electrochemical response is observed. Our spliced DNA walking machine precedes the previous DNA machine in terms of economy and stability due to the strategy of entropy-driven and label-free, and possesses a detection limit to 0.96 pM for detection of ampicillin. Moreover, this DNA machine is expected to detect more analytes by using corresponding aptamer probes. Hence, the entropy-driven DNA walker provides an efficient and practical platform for small molecule analysis.

Colorimetric detection of miRNA-21 by DNAzyme-coupled branched DNA constructs

Fluctuation of nucleic acid expression and ultrasensitive and specific detection of these variations in expression is a crucial subject in molecular medicine and clinical theranostics. A novel DNAzyme-coupled branched hybridization chain reaction (b-HCR) assay is reported for efficient signal-amplified detection of miRNA in this study. This assay was composed of a translator (T) hybridized with miR-21 to initiate the first HCR by hairpin 1 (H1) and hairpin 2 (H2). The primary HCR provided a backbone chain for numerous branches budding through hairpin 3 (H3) and hairpin 4 (H4) assembles. In the presence of hemin, the G-rich domains embedded in H1 and H4 produce an active G-quadruplex DNAzyme upon exposure to a target that could catalyze the oxidation of colorless substrate to colored product. The present approach has the potential to be used for quantitative detection of miR-21 with a sensitivity and a dynamic range of 1 pM and 1 pM to 1 nM, respectively.

Dual amplification in a fluorometric acetamiprid assay by using an aptamer, G-quadruplex/hemin DNAzyme, and graphene quantum dots functionalized with D-penicillamine and histidine.

D-penicillamine and histidine-functionalized graphene quantum dot (DPA-GQD-His) was synthesized and applied in a fluorometric method for determination of acetamiprid using a G-quadruplex DNAzyme. At first DNA probe (probe 1) consists of a target-specific aptamer with two arms of DNA segments. Probe 1 was hybridized with DNA probe 2 composed of a single DNA sequence with two split G-rich DNA sequences. This leads to the formation of a triplex-to-G-quadruplex (TPGQ). Next, acetamiprid was hybridized with the aptamer in the TPGQ to release free DNA probe 2. The released probe 2, in the presence of of K+, undergoes a structural change into a stem-loop structure (by self-complementary hybridization and Hoogsteen hydrogen bonding) that bears a G-quadruplex structure. This is followed by conjugation with hemin to form the G-quadruplex/hemin DNAzyme. The DNAzyme catalyzes the oxidation of o-phenylenediamine by H2O2 to produce a yellow fluorescent product with excitation/emission maxima at 420/560nm. The oxidation product interacts with DPA-GQD-His to achieve a rapid energy transfer between DPA-GQD-His and oxidation product. This increases the fluorescence of the oxidation product and quenches the fluorescence of DPA-GQD-His. DPA-GQD-His also improves the catalytic activity of DNAzyme towards oxidation of ophenylenediamine oxidization and enhances fluorometric response to acetamiprid. The assay works in the 1.0 fM to 1.0nM acetamiprid concentration range and has a 0.38 fM detection limit. It was successfully applied to the determination of acetamiprid in tea. Graphical abstractThe study reported one double amplification strategy for ultrasensitive fluorescence detection of acetamiprid in tea with D-penicillamine and histidine-functionalized graphene quantum dots and G-quadruplex/heminDNAzyme. The analtyical method exhibits ultra high sensitivity, selectivity and rapidity of fluorescenceresponse to acetamiprid.

Hemin-Bridged MOF Interface with Double Amplification of G-Quadruplex Payload and DNAzyme Catalysis: Ultrasensitive Lasting Chemiluminescence MicroRNA Imaging.

Here, we report a double-amplified sensing platform for ultrasensitive chemiluminescence (CL) miRNA detection in real patients' blood in which a hemin-bridged metal-organic framework (MOF) is employed as a functional interface to boost the payload and catalysis of G-quadruplex (G4) DNAzymes. Hemin is here used as the organic ligand for the MOF synthesis, which endows the MOF with an intrinsic peroxidase-like catalytic activity. Most importantly, the MOF surface provides a large amount of binding sites for polymeric G4 DNAzymes that are produced by miRNA-triggered rolling circle amplification reactions, and meanwhile, the interfaced G4 DNAzymes on MOFs (G4/MOFzymes) display an about 100-fold higher catalytic activity than those in solution. By using the G4/MOFzyme catalysts in the luminol/H2O2 CL system, the amplification detection of two acute myocardial infarction (AMI)-related miRNAs (low to 1 fM seen with naked eyes) is achieved in human serum with a smartphone as a portable imaging detector, which provides a facile methodology for point-of-care (POC) diagnosis of AMI. Compared with previous smartphone-based counterparts not requiring sophisticated equipment, this new facile methodology shows both 6 orders of magnitude higher sensitivity and an ∼50-fold longer duration for CL miRNA imaging. These unique features allow our developed G4/MOFzymes to be further employed as a novel luminescent ink for printing commonly used patterns.

Performance of a template enhanced hybridization process in biological media for the detection of a breast cancer biomarker

A split G-quadruplex DNAzyme have been used for analysis of microRNA mimics in biological media with colorimetric assays based on Template enhanced Hybridization Processes, TeHyP.

A signal cascade amplification strategy based on RT-PCR triggering of a G-quadruplex DNAzyme for a novel electrochemical detection of viable Cronobacter sakazakii

Cronobacter sakazakii is an important opportunistic food-borne pathogen, and it can cause severe diseases with main symptoms including neonatal meningitis, necrotizing enterocolitis, and sepsis. For the achievement of practical and convenient detection of viable C. sakazakii, a simple and robust strategy based on the cascade signal amplification of RT-PCR triggered G-quadruplex DNAzyme catalyzed reaction was firstly used to develop an effective and sensitive DNAzyme electrochemical assay. Without viable C. sakazakii in the samples there are no RT-PCR and DNAzyme products, which can cause a weak electrochemical response. Once viable C. sakazakii exists in the samples, an obvious enhancement of the electrochemical response can be achieved after the target signal is amplified by RT-PCR and the resulting DNAzyme, which catalyze the oxidation of 3,3',5,5'-tetramethylbenzidine (TMB) by H2O2 with the assistance of the cofactor hemin. Our novel assay can be performed in a range of 2.4 × 107 CFU mL-1 to 3.84 × 104 CFU mL-1 (R2 = 0.9863), with a detection limit of 5.01 × 102 CFU mL-1. Through the assay of 15 real samples, electrochemical detection assay provided the same results as conventional detection methods. Therefore, detection of viable C. sakazakii based on G-quadruplex DNAzyme electrochemical assay with RT-PCR demonstrates the significant advantages of high sensitivity, low cost and simple manipulation over existing approaches and offers an opportunity for potential application in pathogen detection.