Integrated Photoelectrochemical-SERS Platform Based on Plasmonic Metal-Semiconductor Heterostructures for Multidimensional Charge Transfer Analysis and Enhanced Patulin Detection.

Comprehending the charge transfer mechanism at the semiconductor interfaces is crucial for enhancing the electronic and optical performance of sensing devices. Yet, relying solely on single signal acquisition methods at the interface hinders a comprehensive understanding of the charge transfer under optical excitation. Herein, we present an integrated photoelectrochemical surface-enhanced Raman spectroscopy (PEC-SERS) platform based on quantum dots/metal-organic framework (CdTe/Yb-TCPP) nanocomposites for investigating the charge transfer mechanism under photoexcitation in multiple dimensions. This integrated platform allows simultaneous PEC and SERS measurements with a 532 nm laser. The obtained photocurrent and Raman spectra of the CdTe/Yb-TCPP nanocomposites are simultaneously influenced by variable bias voltages, and the correlation between them enables us to predict the charge transfer pathway. Moreover, we integrate gold nanorods (Au NRs) into the PEC-SERS system by using magnetic separation and DNA biometrics to construct a biosensor for patulin detection. This biosensor demonstrates the voltage-driven ON/OFF switching of PEC and SERS signals, a phenomenon attributed to the plasmon resonance effect of Au NRs at different voltages, thereby influencing charge transfer. The detection of patulin in apples verified the applicability of the biosensor. The study offers an efficient approach to understanding semiconductor-metal interfaces and presents a new avenue for designing high-performance biosensors.

Wavelength-Resolved Janus Biosensing Interface for Ratiometric Electrochemical Analysis.

The Janus interface, comprising multiple functional heterointerfaces with contrasting functionalities within a single interface, has recently garnered widespread research interest. Herein, a Janus biosensing interface is obtained via wavelength-resolved laser illumination. Deoxyribonucleic acid bridges the electrochemical probe of methylene blue (MB) and plasmonic gold nanoparticles (AuNPs), achieving a sensitive detection performance. MB shows differential electrochemical signals under front (I532front) and back (I650back) laser illumination at 532 and 650 nm, respectively, owing to the selective wavelength-resolved effect. Thus, the presence of a wavelength-resolved laser enabled the design of a biosensing interface with Janus properties. The change in the distance between MB and AuNPs induced by aflatoxin B1 (AFB1) indicates that a sensitive response of the Janus biosensing interface can be achieved. A ratiometric strategy is introduced to describe the electrochemical signals of the I532front and I650back for improved robustness. The obtained linear range is 0.0005-50 ng mL-1, with a detection limit of 0.175 pg mL-1. Our study demonstrated that the wavelength-resolved Janus interface enables an electrochemical biosensor with excellent sensitivity. This finding provides an efficient approach for improving biosensor performance.

Photoelectrochemical aptasensing with methylene blue filled Ni-MOFs nanocomposite by spatial confinement for microcystin-LR detection.

Microcystin LR (MC-LR) is a hazardous cyanotoxin produced by cyanobacteria during freshwater eutrophication, which can cause liver cancer. Here, a photoelectrochemical (PEC) aptasensor based on methylene blue (MB)-loaded Ni-MOF composite (Ni-MOF/MB) with spatial confinement was constructed for the sensitive detection of MC-LR. Ni-MOF with two-dimensional sheet structure was prepared via a liquid-liquid interface synthesis method with environmental-friendly solvent and milder reaction conditions. Benefiting from the uniform pore size, Ni-MOF acted as reaction platform to anchor the photosensitive molecule MB. The electron donor, ascorbic acid (AA), was produced by alkaline phosphatase (ALP) loaded on DNA strand catalyzing ascorbic acid phosphate. The generated AA was absorbed by Ni-MOF/MB, thereby effectively improving the utilization of AA and avoiding the external environment interferences to enlarge the photocurrent of MB. For analysis, ALP-labeled aptamer can specifically recognize MC-LR by forming a complex to strip from aptasensor, thus leading to a  decreased photocurrent. The developed PEC aptasensor offered a linear range of 10 fM-100 pM with a detection limit of 6 fM. It was successfully employed for detecting MC-LR in farm water and fish meat, and the results were validated by ultrahigh-performance liquid chromatography-mass spectrometry. This method presents a new idea of MOF-limited domain for PEC aptasensing.

Split aptamer-based sandwich-type ratiometric biosensor for dual-modal photoelectrochemical and electrochemical detection of 17ß-estradiol.

BACKGROUND: As one of the most potent environmental estrogens, 17ß-estradiol (E2), which can be enriched into organisms through the food chain and cause harmful biological effects in humans, has been frequently detected in the water environment of the world. High performance liquid chromatography (HPLC) and gas chromatograohy-mass spectrometry (GC/MS) have been widely used for quantification of E2. Despite excellent accuracy, tedious pretreatment and expensive instruments result in their limited application. It is clear that there is an urgent need to establish simple, sensitive and accurate methods for the determination of E2. RESULTS: A split aptamer-based sandwich-type ratiometric biosensor based on split aptamer was developed by coupling photoelectrochemical and electrochemical assays for E2 detection. For analysis, the two fragments of split aptamer recognized E2 by forming sandwich structure, which triggered hybridization chain reaction (HCR) to produce double-stranded DNA (dsDNA) with CdTe quantum dots (QDs) labeled hairpin DNA. The resultant dsDNA can further absorb methylene blue (MB) to sensitize CdTe QDs for an enlarged photocurrent (IPEC) and output a redox current of IMB, and both of them acted as response signals for detection; [Fe(CN)6]3-/4- probe produced redox current of I[Fe(CN)6]3-/4- as reference signal. Using IMB/I[Fe(CN)6]3-/4- and IPEC/I[Fe(CN)6]3-/4- as yardsticks, the developed split aptamer-based sandwich-type ratiometric biosensor provides two linear ranges of 0.1-5000 pg mL-1 for IMB/I[Fe(CN)6]3-/4- and 0.1-10000 pg mL-1 for IPEC/I[Fe(CN)6]3-/4- with detection limits of 0.06 pg mL-1 and 0.02 pg mL-1, respectively. SIGNIFICANCE: These results of the biosensor are benefiting from the coupling of photoelectrochemical (PEC) and electrochemical (EC) assays as well as the unique cooperative recognition mechanism of split aptamer. This method not only enabled the biosensor to be successfully applied to the determination of E2 in lake water, but also broadens the prospects for the realization of sensitive and accurate detection of E2.

All-in-one fabrication of a ratiometric electrochemical aptasensor with tetrahedral DNA nanostructure for fumonisin B1 detection.

Here, we develop an all-in-one strategy for efficient assembly of an electrochemical aptasensor. A multifunctional structure based on a tetrahedral DNA nanostructure (TDN) was synthesized via a one-step annealing process, providing DNA fixation, target recognition, signal amplification and space regulation. Based on the integration of this multifunctional structure, the sensing interface was assembled in one step. A ratiometric aptasensor was constructed by anchoring methylene blue (MB) to the TDN and ferrocene (Fc) on the cDNA. Using the ratio of the currents obtained from Fc and MB as a measure, the developed aptasensor shows excellent analytical performance for fumonisin B1 detection. This strategy is universal and could simplify the fabrication of aptasensors.

Portable Visual Photoelectrochemical Biosensor Based on a MgTi2O5/CdSe Heterojunction and Reversible Electrochromic Supercapacitor for Dual-Modal Cry1Ab Protein Detection.

Reversible electrochromic supercapacitors (ESCs) have attracted considerable interest as visual display screens. The use of ESCs in combination with a photoelectrochemical (PEC) biosensor promises to improve the detection efficiency. Herein, a visual PEC biosensor is developed by introducing a circuit module between a PEC-sensing platform (PSP) and a reversible ESC for Cry1Ab protein detection. In PSP, a type II MgTi2O5/CdSe heterojunction effectively drives charge separation by their cross-matched band gap structures, generating an amplified photocurrent. Next, the circuit module is designed to connect the PSP and ESC, realizing the signal conversion from photocurrent to voltage. ESC, as a visual display screen, produces reversible color changes with different voltages. As the concentration of Cry1Ab increases, the photocurrent decreases due to the specific binding between the aptamer and Cry1Ab in PSP, while the color of the reversible ESC changes from green to blue. To improve the integrity of the device, a portable PEC biosensor is further constructed via three-dimensional printing for dual-modal Cry1Ab protein detection, thus collecting both PEC and visual signals. The linear ranges are 0.3-3000 ng mL-1 for PEC mode and 1-1000 ng mL-1 for visual mode. This work presents a portable, efficient, sensitive, and visualized detection system, providing an important reference for practical visualization applications.

Space-Confined Electrochemical Aptasensing with Conductive Hydrogels for Enhanced Applicability to Aflatoxin B1 Detection.

Aflatoxin B1 (AFB1) contamination has received considerable attention for the serious harm it causes and its wide distribution. Hence, its efficient monitoring is of great importance. Herein, a space-confined electrochemical aptasensor for AFB1 detection is developed using a conductive hydrogel. Plasmonic gold nanoparticles (AuNPs) and methylene blue-embedded double-stranded DNA (MB-dsDNA) were integrated into the conductive Au-hydrogel by ultraviolet (UV) polymerization. Specific recognition of AFB1 by the aptamer released MB from MB-dsDNA in the matrix. The free DNA migrated to the outer layer due to electrostatic repulsion during the Au-hydrogel formation. The electrochemical aptasensor based on this Au-hydrogel offered a twofold enlarged oxidation current of MB (IMB) compared with that recorded in the homogeneous solution for AFB1 detection. Upon light illumination, this IMB was further enlarged by the local surface plasmon resonance (LSPR) of the AuNPs. Ultimately, the Au-hydrogel-based electrochemical aptasensor provided a detection limit of 0.0008 ng mL-1 and a linear range of 0.001-1000 ng mL-1 under illumination for AFB1 detection. The Au-hydrogel allowed for space-confined aptasensing, favorable conductivity, and LSPR enhancement for better sensitivity. It significantly enhanced the applicability of the electrochemical aptasensor by avoiding complicated electrode fabrication and signal loss in a bulk homogeneous solution.

Electric field-enabled aptasensing interfacial engineering to simultaneously enhance specific preconcentration and electrochemical detection of mercury and lead ions.

Aptamers with strong affinity to heavy metal ions (HMIs) allow fabrication of electrochemical sensors with high selectivity and sensitivity, while controllable regulation of aptamer-HMI recognition at the sensing interface, which is vital for better analytical performance, remains challenging. Here, an electric field-based strategy for engineering an aptasensing interface was proposed to realize the specific preconcentration and accurate detection of mercury (Hg2+) and lead (Pb2+) ions with a ratiometric electrochemical sensor. The working principle is to apply an electric field to drive HMIs to approach the aptamer and retain the orientation of the DNA structure. Anthraquinone-2-carboxylic acid (AQ)-labeled complementary DNA was designed to simultaneously bind a ferrocene (Fc)-labeled aptamer for Hg2+ and a methylene blue (MB)-labeled aptamer for Pb2+, and the sensing interface was fabricated with this presynthesized DNA structure. For preconcentration, an electric field of 3.0 V pushed HMIs to approach the aptamer and retained the orientation of DNA to favor the following recognition; for detection, the oriented DNA in 2.5 V electric field offered a stable current of AQ as a reference. In this way, currents of AQ, Fc and MB were used to produce ratiometric signals of IAQ/IFc and IAQ/IMB for Hg2+ and Pb2+, respectively. Such a strategy allowed the simultaneous detection of Hg2+ and Pb2+ within 30 min with detection limits of 0.69 pM and 0.093 pM, respectively. The aptasensor was applied for soil, water, and crayfish analysis in paddy fields. The electric field-enabled strategy offers a new way to fabricate high-performance electrochemical aptasensor for HMIs detection.

Photo-enhanced electrochemical and colorimetric dual-modal aptasensing for aflatoxin B1 detection based on graphene-gold Schottky contact.

A photo-enhanced electrochemical (PEEC) and colorimetric (CM) dual-modal aptasensor was developed with rGO-AuNP Schottky contact for AFB1 monitoring. The PEEC mode allowed the ultrasensitive quantitation based on the photo-enhanced electroactivity mechanism, while the CM mode offered a rapid threshold-level qualitative assay with a portable colorimeter.

In-depth interpretation of aptamer-based sensing on electrode: Dual-mode electrochemical-photoelectrochemical sensor for the ratiometric detection of patulin.

Electrochemical aptasensors have been extensively used to quantify food contaminants (e.g., mycotoxin) by using high-affinity aptamer for target recognition. Yet, analytical performance of aptasensors using different aptamers can be varied for the same target. Here, four aptamers with different sequences (i.e., A22, A34, A42, and A45) of patulin (PAT) were selected to estimate sensing behaviors at electrodes with electrochemical (EC) and photoelectrochemical (PEC) assays. Synergistic effect of steric hindrance and electron transfer distance was found to significantly affect EC and PEC response for PAT at aptasensors fabricated with A22, A34, A42, or A45. Eventually, A22 emerged to be the optimal aptamer for aptasensing, despite the highest affinity of A42 to PAT. The A22-based EC-PEC dual-mode ratiometric aptasensor offered a linear range of 50 fg mL-1 - 500 ng mL-1 with a detection limit of 30 fg mL-1 for PAT, and it was applied to apple product (i.e., juice, puree) analysis.

Photoelectrochemical and visual dual-mode sensor for efficient detection of Cry1Ab protein based on the proximity hybridization driven specific desorption of multifunctional probe.

Currently, the development of sensitive and visual strategy for Cry1Ab detection, particularly using a switchable dual-mode detection system based on a single component, remains a great challenge. Here, a photoelectrochemical (PEC) and visual dual-mode sensor was designed for Cry1Ab detection based on a proximity hybridization driven multifunctional probe. In the presence of Cry1Ab, specific desorption of the antibody-DNA conjugate was achieved via sufficient proximity hybridization, leading to the selective release of the multifunctional signal probe, i.e., antibody-labeled single-stranded DNA-gold nanoparticles (Ab1-S1-AuNPs). The released Ab1-S1-AuNPs reduced the photocurrent signal and produced a colored response, thereby achieving PEC and visual dual-mode detection based on a single component. Owing to the different signal generation mechanisms, two independent signals were obtained simultaneously, which provided self-verification to improve reliability and accuracy. Taking advantage of the PEC sensitive detection and visual prediction, the dual-mode sensor achieved efficient detection of the Cry1Ab protein. The developed sensor was successfully used to determine Cry1Ab in corn, wheat, and soil samples with satisfactory results. This method offers a promising biosensing platform for the on-site detection of Cry1Ab protein.

Engineering the Signal Transduction between CdTe and CdSe Quantum Dots for in Situ Ratiometric Photoelectrochemical Immunoassay of Cry1Ab Protein.

Controllable modulation of a response mode is extremely attracting to fabricate biosensor with programmable analytical performances. Here, we reported a proof-of-concept ratiometric photoelectrochemical (PEC) immunoassay of Cry1Ab protein based on the signal transduction regulation at the sensing interface. A sandwich-type PEC structure was designed so that gold nanorods sensitized quantum dots to fix primary antibody (Au NRs/QDs-Ab1) and methylene blue sensitized QDs to combine a second antibody (MB/QDs-Ab2), which served as photoelectric substrate and signal amplifier, respectively. Unlike common recognition element, such a sandwich-type PEC structure allowed for the in situ generation of two specific response signals. For analysis, Cry1Ab captured by Au NRs/QDs-Ab1 led to a decreased photocurrent (ICry1Ab), while the subsequently anchored MB/QDs-Ab2 produced another photocurrent (IMB). Noteworthy, by taking advantage of the different energy band gaps of QDs, varying locations of CdTe and CdSe QDs could realize different signal transduction strategies (i.e., Mode 1 and Mode 2). Investigations on data analysis of ICry1Ab and IMB via different routes demonstrated the superior analytical performances of ratiometry (Mode 1). Consequently, the ratiometric PEC immunosensor offered a linear range of 0.01-100 ng mL-1 with a detection limit of 1.4 pg mL-1. This work provides an efficient strategy for in situ collection of multiple photocurrents to design ratiometric PEC sensors.

Signal-enhanced strategy for ratiometric aptasensing of aflatoxin B1: Plasmon-modulated competition between photoelectrochemistry-driven and electrochemistry-driven redox of methylene blue.

Efficient signal amplification using light irradiation in electrochemistry-based sensing attracts growing interest; however, the modulation of electrochemical and photoelectrochemical behaviors of bifunctional probes remains challenging for the better analytical performance. Here, we found that the bifunctional probe methylene blue (MB) under irradiation followed two routes, i.e., photoelectrochemistry (PEC)-driven and electrochemistry (EC)-driven route, while plasmon-modulated competition between PEC-driven and EC-driven routes efficiently enhanced the analytical performance of aptasensor. Ferrocene-labeled aptamer (Fc-apt) was applied to tune the interaction between MB labeled complementary DNA (MB-cDNA) and plasmonic gold nanoparticles (AuNPs). Under visible light illumination, the aptasensor output a weak current of MB (IMB) for the depressed EC-driven redox. The binding of Fc-apt with target led to its stripping from electrode and released MB-cDNA to approach AuNPs, and an increase in IMB was observed due to the synergy of the depressed PEC-driven redox while the restored EC-driven redox. In this way, the amplified response for target could be obtained by depressing background signal while enlarging response signal of IMB under visible light. Consequently, the developed model ratiometric aptasensor offered a higher sensitivity for the determination of aflatoxin B1. Our observation should be favorable to shed light on photo-induced electrochemical mechanism, and the proposed amplification strategy is valuable for the construction of efficient sensors.

Electric Field-Induced Specific Preconcentration to Enhance DNA-Based Electrochemical Sensing of Hg2+ via the Synergy of Enrichment and Self-Cleaning.

Efficient preconcentration is critical for sensitive and selective electrochemical detection of metal ions, but rapid specific enrichment with depressed absorption of interfering ions at the electrode is challenging. Here, we proposed an electric field-induced specific preconcentration to boost the analytical performance of DNA-based electrochemical sensors for Hg2+ detection. As for such preconcentration, a positive external electric field was first used to enrich Hg2+ at an electrode assembled with T-rich DNA, thus boosting T-Hg2+-T recognitions. The following applied inverse electric field strips the nonspecifically absorbed Hg2+ and other interfering ions, thus depressing matrix interferences via self-cleaning. Based on this principle, we designed a portable device to realize programmable control of electric fields; a T-Hg2+-T recognition-based electrochemical sensor was thus fabricated as a model platform to assess the feasibility of electric field-induced preconcentration. The experimental results revealed that such a strategy decreased the time of T-Hg2+-T-based recognition from 60 to 20 min and led to detection with better reproducibility by depressing the influence of free Hg2+ as well as interfering ions. This strategy offered Hg2+ detection limits of 0.01 pM─three-fold better than that without preconcentration─within 22 min. The proposed preconcentration strategy offers a new way to enhance the analytical performance of sensing at the solid-liquid interface.

Temperature and pH tolerance ratiometric aptasensor: Efficiently self-calibrating electrochemical detection of aflatoxin B1.

Ratiometric electrochemical aptasensors based on the two different redox reporters can hardly achieve the efficient self-calibration, as a result of variations between reporters. In this work, we reported a ratiometric electrochemical aptasensor using ferrocene as a single redox reporter (Fc) to achieve self-calibrating electrochemical detection of aflatoxin B1 (AFB1). The hybrid duplex of Fc-labeled AFB1 aptamer (Fc-Apt) and Fc-labeled assistant DNA (Fc-aDNA) was designed to construct the sensing interface. Such DNA structure adopted the fixed molar ratio (1:1) and locations (the distances to electrode) of these two types of Fc (i.e., Fc-Apt and Fc-aDNA). In this way, the ratio of redox currents from Fc-Apt (IFc-Apt) and Fc-aDNA (IFc-aDNA) kept stable under the varied temperatures (15-40 °C) and pH values (4-10). Moreover, values of IFc-Apt and IFc-aDNA under the different conditions can be calculated simply by recording the total current of Fc (IFc-total). For analysis, the recognition of AFB1 by Fc-Apt led to the striping of their complex from the aptasensor, resulting in a decrease in IFc-Apt while IFc-aDNA remained stable. Consequently, the developed aptasensor using the ratio of IFc-Apt/IFc-aDNA as a yardstick offered a linear range of 0.1-10000 pg mL-1 with a detection limit of 0.012 pg mL-1 for the detection of AFB1. The applicability of aptasensor was validated by corn sample analysis, which exhibited comparable reliability and accuracy of gold standard method, i.e., HPLC-MS/MS. Our work provided an efficient strategy to fabricate high-performance ratiometric electrochemical aptasensors.

Dual-ratiometric electrochemical aptasensor enabled by programmable dynamic range: Application for threshold-based detection of aflatoxin B1.

Aptamer-based sensor with high-specificity typically has a fixed linear range due to the host-guest binding. To expand the dynamic range for multiple scenarios, we report here a dual-ratiometric electrochemical aptasensor that integrates two aptamer profiles with varied affinity into a single sensing interface for aflatoxin B1 (AFB1) detection. Using functional aptamer as recognition element and generator of signals, we fabricate the dual-ratiometric aptasensor with anthraquinone loaded reduced graphene oxide (AQ-rGO), methylene blue labeled hairpin DNA (MB-DNA), and ferrocene labeled linear aptamer (Fc-apt), using MB and Fc probes to work in tandem while AQ as reference. The current of Fc (IFc) is used to detect the low-concentration analyte, and that of MB (IMB) varies when the concentration of AFB1 reaches a threshold. The threshold switch for IMB could be tuned by engineering the quantity ratio of Fc-apt and MB-DNA (a ratio of 1:1 is used here). The aptasensor can rapidly achieve the positive (+)/negative (-) detection of AFB1 with a threshold concentration of 10 pg mL-1, while the quantitative detection employs the ratio of current of AQ, MB, and Fc (i.e., IAQ/IMB and IAQ/IFc) as yardsticks, offering two linear ranges of 10-106 and 10-2-5 × 104 pg mL-1, respectively. The aptasensor is successfully used to monitor the amount of AFB1 in a 7-days mildew process of peanut. Such a protocol opens a new way for the design of sensors with the programmable linear range in the advanced biological and chemical sensing.

Regulation of Ru(bpy)32+ Electrochemiluminescence Based on Distance-Dependent Electron Transfer of Ferrocene for Dual-Signal Readout Detection of Aflatoxin B1 with High Sensitivity.

Ferrocene (Fc) is a common quencher of Ru(bpy)32+ luminescence. However, interactions between Fc and Ru(bpy)32+ can be extremely complicated. In this work, we reported the first use of Fc to regulate the electrochemiluminescence (ECL) of Ru(bpy)32+ by tuning the length of the DNA sequence between Fc and the luminophore of nitrogen-doped graphene quantum dots-Ru(bpy)32+-doped silica nanoparticles (SiO2@Ru-NGQDs). The ECL of SiO2@Ru-NGQDs was depressed when the distance between Ru(bpy)32+ and Fc was less than 8 nm; a stronger ECL was observed when the distance was more than 12 nm. The switching of the ECL of Ru(bpy)32+ by Fc was attributed to the electron transfer mechanism, in which Fc participated in the redox of Ru(bpy)32+ for "signal-off" ECL; this favored electron transfer at the electrode fabricated with an Fc-labeled aptamer (Fc-apt) and SiO2@Ru-NGQDs for "signal-on" ECL depending on the length of the DNA sequence. Here, a dual-signal readout aptasensor for aflatoxin B1 (AFB1) detection was developed via the enhanced ECL of SiO2@Ru-NGQDs by Fc-apt. The redox currents of Fc and the ECL of Ru(bpy)32+ were simultaneously collected as yardsticks, and both decreased with higher concentrations of AFB1. The aptasensor allowed linear ranges of 3 × 10-5 to 1 × 102 ng mL-1 for ECL mode and 1 × 10-3 to 3 × 103 ng mL-1 for electrochemical mode. Our work provides insight into the interactions between Fc and Ru(bpy)32+. The dual-signal readout strategy is a potential platform for the versatile design of aptasensors.

Interaction between the functionalized probes: The depressed efficiency of dual-amplification strategy on ratiometric electrochemical aptasensor for aflatoxin B1.

Signal amplification is one of the most effective ways to develop the high-performance electrochemical sensors. However, it can be more complicated for ratiometric detections. Herein, a ratiometric electrochemical aptasensor for aflatoxin B1 (AFB1) was proposed by taking advantage of a dual-amplification strategy by coupling of DNA walker (DW) with hybridization chain reaction (HCR). The special binding of AFB1 with ferrocene (Fc)-labelled aptamer triggers DW on hairpin DNA (hDNA) tracks to produce abundant double-stranded DNA (dsDNA). HCR-based strand amplification occurs on these dsDNA to absorb more methylene blue (MB). Then current ratio of MB (IMB) and Fc (IFc) is designed as a yardstick to detect AFB1. Our experiments reveal that the interaction between Fc and MB (i.e., steric hindrance, electron mediator) varies. In addition to steric hindrance, the presence of MB also acts as electron mediator, thereby facilitating the electron transfer between Fc and electrode. Such combined effect consequently depresses the efficiency of dual-amplification strategy to improve the detection. The developed ratiometric electrochemical aptasensor allows the accurate detection of AFB1 in the 0.003-3 pg mL-1 range. Our work has shed light on the amplification strategy for ratiometric sensing, and provided a new route in integrating different amplification strategies.