RESUMO
We developed an axis-mode donor-DNA-acceptor electrochemical system to distinguish whether electron transfer in DNA occurs by tunneling or hopping. In the axis-mode, rigid stem-loop DNA was designed with the redox probe Ag+ embedded at the axis of the strand through a C-Ag+-C mismatch, which was immobilized onto the electrode surface in a saturated manner. Thus, the rotation, swing, and bending of the DNA strand were restricted and then the number of Ag+, the distance L between Ag+ and the electrode, and the chemical environment could be precisely controlled. In addition, fast scan cyclic voltammetry was applied to realize the in situ redox reaction of Ag+, without diffusion away from the electrode and the ensuing deconstruction of the stem-loop DNA. In this case, as a direct indicator of rate, the peak Faradaic current ip was extracted and used to fit the tunneling mechanism i â exp (-ßL) and the hopping mechanism i â L-η. The value of ß was determined to be 0.100 Å-1, which is consistent with the range of 0.1â¼1.5 Å-1 reported previously, while η was determined to be 0.677, which is completely beyond the correct range of 1 ≤ η ≤ 2, demonstrating that electron transfer in DNA occurs by tunneling instead of hopping or that tunneling dominates. Additionally, current additivity and the irrelevance of the base sequence illustrate this point again. Thus, the possibility of independent parallel tunneling currents in DNA strands is revealed, which is helpful for recognizing the feasibility of DNA-based wires and devices.
Assuntos
DNA , Elétrons , Transporte de Elétrons , Oxirredução , EletrodosRESUMO
An electrochemical aptasensor for ultrasensitive detection of Staphylococcus aureus (SA) has been developed based on stepwise signal amplification. In the sample processing stage, the specific recognition between SA and aptamer triggers the enzyme-assisted cyclic cleavage to produce a large amount of target DNA (tDNA), realizing the first-level signal amplification. In the sensor assembly stage, tDNA induces a catalytic hairpin assembly (CHA) cycle to capture much more hairpin DNA H2 labeled by the electrochemical tag ferrocene, bringing the second-level signal amplification. In the signal detection stage, ferrocene is quasi-adsorbed on the electrode surface, and a high redox peak current linearly increasing with the scan rate up to 1000 V/s has been obtained by fast scan cyclic voltammetry (FSCV), achieving the third-level signal amplification. Under the optimized experimental conditions, the linear range and detection limit are 1 ~ 108 CFU/mL and 0.3 CFU/mL, respectively. The sensor has good reproducibility, stability, and sensitivity, affording practical sample detection. This detection principle is widely applicable to other pathogens, and provides a new path for the establishment of highly sensitive detection strategies.
Assuntos
Aptâmeros de Nucleotídeos , Técnicas Biossensoriais , DNA , Técnicas Eletroquímicas , Ouro , Metalocenos , Reprodutibilidade dos Testes , Staphylococcus aureusRESUMO
A clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12a-mediated dual-mode electrochemical biosensor without polymerase chain reaction (PCR) amplification was designed for sensitive and reliable detection of genetically modified soybean SHZD32-1. A functionalized composite bionanomaterial Fe3O4@AuNPs/DNA-Fc&Ru was synthesized as the signal unit, while a characteristic gene fragment of SHZD32-1 was chosen as the target DNA (tDNA). When Cas12a, crRNA, and tDNA were present simultaneously, a ternary complex Cas12a-crRNA-tDNA was formed, and the nonspecific cleavage ability of the CRISPR/Cas12a system toward single-stranded DNA was activated. Thus, the single-stranded DNA-Fc in the signal unit was cleaved, resulting in the decrease in the fast scan voltammetric (FSV) signal from ferrocene (Fc) and the increase in the electrochemiluminescence (ECL) signal from ruthenium complex (Ru) inhibited by Fc. The linear range was 1-107 fmol/L for ECL and 10-108 fmol/L for FSV, and the limit of detection (LOD) was 0.3 fmol/L for ECL and 3 fmol/L for FSV. Accuracy, precision, stability, selectivity, and reliability were all satisfied. In addition, PCR-free detection could be completed in an hour at room temperature without requiring complicated operation and sample processing, showing great potential in the field detection of genetically modified crops.
Assuntos
Técnicas Biossensoriais , Nanopartículas Metálicas , Sistemas CRISPR-Cas/genética , Produtos Agrícolas , Ouro , Plantas Geneticamente Modificadas/genética , Reação em Cadeia da Polimerase , Reprodutibilidade dos Testes , Glycine max/genéticaRESUMO
In this study, a low field nuclear magnetic resonance (LF-NMR) homogeneous sensor was constructed for detection of Escherichia coli (E. coli) based on the copper metabolism of E. coli triggered click reaction. When live E. coli was present, a large amount of Cu2+ ions were transformed into Cu+ via copper metabolism, which then catalyzed a Cu+-catalyzed azide-alkyne cycloaddition (CuAAC) reaction between two materials, azide group modified gadolinium oxide nanorods (Gd2O3-Az) and PA-GO@Fe3O4 i.e., graphene oxide (GO) loaded with large amounts of alkynyl (PA) groups and Fe3O4 nanoparticles simultaneously. After magnetic separation, unbound Gd2O3-Az was dissolved by added hydrochloric acid (HCl) to generate homogeneous Gd3+ solution, enabling homogeneous detection of E. coli. Triple signal amplification was achieved through the CuAAC reaction induced by E. coli copper metabolism, functional nanomaterials, and HCl assisted homogeneous detection. Under the optimal experimental conditions, the linear range and limit of detection (LOD) for E. coli were 10-1.0 × 107 CFU/mL and 3.5 CFU/mL, respectively, and the relative standard deviations (RSDs) were all less than 2.8 %. In addition, the sensor has satisfactory selectivity, stability and practical sample application capability, providing a new approach for the LF-NMR detection of food-borne pathogenic bacteria.
Assuntos
Alcinos , Azidas , Química Click , Cobre , Escherichia coli , Grafite , Escherichia coli/isolamento & purificação , Cobre/química , Azidas/química , Grafite/química , Alcinos/química , Espectroscopia de Ressonância Magnética , Reação de Cicloadição , Gadolínio/química , Limite de Detecção , Nanotubos/químicaRESUMO
Herein, an electrochemical sensor for single-cell determination of bacteria was developed based on target-triggered click chemistry and fast scan voltammetry (FSV). In it, bacteria not only are the detection target, but also can use their own metabolism to achieve first-level signal amplification. More electrochemical labels were immobilized on functionalized 2D nanomaterials to achieve second-level signal amplification. At 400 V/s, FSV can achieve third-level signal amplification. The linear range and limit of quantification (LOQ) are 1 â¼ 108 CFU/mL and 1 CFU/mL, respectively. When the reaction time of E. coli-instructed Cu2+ reduction is extended to 120 min, PCR-free single-cell determination of E. coli was achieved by electrochemical method first time. The feasibility of the sensor was verified by analysis of E. coli in seawater and milk samples with recoveries ranging from 94% to 110%. This detection principle is widely applicable, providing a new path for the establishment of single-cell detection strategy for bacteria.
Assuntos
Técnicas Biossensoriais , Escherichia coli , Química Click/métodos , Técnicas Biossensoriais/métodos , Ouro , Técnicas Eletroquímicas/métodos , Limite de DetecçãoRESUMO
Herein, a Faraday cage-type electrochemiluminescence biosensor was designed for the detection of human breast cancer cell MCF-7. Two kinds of nanomaterials, Fe3O4-APTs and GO@PTCA-APTs, were synthesized as capture unit and signal unit, respectively. In presence of the target MCF-7, the Faraday cage-type electrochemiluminescence biosensor was constructed by forming a complex "capture unit-MCF-7-signal unit". In this case, lots of electrochemiluminescence signal probes were assembled and could participate in the electrode reaction, achieving a significant increase in sensitivity. In addition, the double aptamer recognition strategy was adopted to improve the capture, enrichment efficiency and detection reliability. Under optimal experimental conditions, the limit of detection was 3 cells/mL. And, the sensor could afford the detection of actual human blood samples, which is the first report on the detection of intact circulating tumor cells by the Faraday cage-type electrochemiluminescence biosensor.
Assuntos
Técnicas Biossensoriais , Células Neoplásicas Circulantes , Humanos , Técnicas Eletroquímicas , Células MCF-7 , Reprodutibilidade dos Testes , Medições Luminescentes , Limite de DetecçãoRESUMO
In this work, a novel fast scan digital circuit for voltammetric analysis with precious ohmic drop compensation is developed, which is achieved through online measuring solution resistance first and then proportionally feedbacking the output signal to potentiostat's in-phase input through a potentiometer. It mainly consists of a solution resistance measurement module based on AD5933 chip, an ohmic drop automatic compensation module and a STM32F103ZET6 microcontroller. The performance of the circuit is checked successively using pure resistances, RC dummy cells, RC dummy cells incorporating a pseudo-faradaic component, and the ferrocene redox system. Results show that, precise ohmic drop compensation can be realized online and automatically, affording fast scan cyclic voltammetric (FSCV) analysis for theoretical electrochemical cells at 2000 V/s and that for practical electrochemical system using conventional electrodes at 1600 V/s. Based on this circuit, a very simple DNA biosensor for ultrasensitive detection of mercuric ions was explored. Benefitting from the high sensitivity brought by the high scan rate, the limit of quantitation (LOQ) can reach 1 pmol/L, demonstrating the application potential of FSCV in the field of ultrasensitive electrochemical detection.