Ratiometric Aptasensor Research Articles

Ratiometric Fluorescence Aptasensor of Allergen Protein Based on Multivalent Aptamer-Encoded DNA Flowers as Fluorescence Resonance Energy Transfer Platform.

Life-threatening allergic reactions to food allergens, particularly peanut protein Ara h1, are a growing public health concern affecting millions of people worldwide. Thus, accurate and rapid detection is necessary for allergen labeling and dietary guidance and ultimately preventing allergic incidents. Herein, we present a novel ratiometric fluorescence aptasensor based on multivalent aptamer-encoded DNA flowers (Mul-DNFs) for the high-stability and sensitive detection of allergen Ara h1. The flower-shaped Mul-DNFs were spontaneously packaged using ultralong polymeric DNA amplicons driven by a rolling circle amplification reaction, which contains a large number of Ara h1 specific recognition units and has excellent binding properties. Furthermore, dual-color fluorescence-labeled Mul-DNFs probes were developed by hybridizing them with Cy3- and Cy5-labeled complementary DNA (cDNA) to serve as a ratiometric fluorescence aptasensor platform based on fluorescence resonance energy transfer. Benefiting from the combined merits of the extraordinary synergistic multivalent binding ability of Mul-DNFs, the excellent specificity of the aptamer, and the sensitivity of the ratiometric sensor to avoid exogenous interference. The developed ratiometric aptasensor showed excellent linearity (0.05-2000 ng mL-1) with a limit of detection of 0.02 ng mL-1. Additionally, the developed ratiometric fluorescence aptasensor was utilized for quantifying the presence of Ara h1 in milk, infant milk powder, cookies, bread, and chocolate with recoveries of 95.7-106.3%. The proposed ratiometric aptasensor is expected to be a prospective universal aptasensor platform for the rapid, sensitive, and accurate determination of food and environmental hazards.

A novel “off–on” ratiometric aptasensor based on dual-emissive persistent luminescent nanoparticles for the detection of dopamine

Dopamine, a neurotransmitter with multiple functions in the brain. The abnormal of dopamine levels are generally considered to be associated with various nervous system diseases. Herein, a novel ‘‘off–on” single-excited dual-emissive ratio luminescent aptasensor for highly sensitive and accurate monitoring of DA levels has been prepared via electrostatic interaction connecting persistent luminescent nanoparticles (PLNPs) ZnGaO:Sm and ZnGeO:Mn with different emission wavelengths. ZnGaO:Sm@Cy5.5-ZnGeO:Mn (ZGS@Cy5.5-ZGM), this ratiometric aptasensor exhibits two-emission peaks at 533 and 700 nm under a single-excitation wavelength of 254 nm. The afterglow luminescence of ZnGaO:Sm-apt@Cy5.5 at 700 nm is effective quenched by complementary chains of Cy5.5-modified DA aptamers with 710 nm specific absorption and only efficiently recovered by DA; meanwhile, the afterglow luminescence of ZnGeO:Mn as reference at 533 nm remains unchanged. The as-designed autofluorescence-free and exogenous-free interference ratiometric sensing system ZGS@Cy5.5-ZGM showed high selectivity for DA in complex biological samples. Under optimal detection conditions, the luminescence intensity ratio Δ(I700/I533) showed a good linear relationship with the concentration of DA in the range of 0.02–80 μM and the detection limit as low as 4 nM. The recoveries of DA in serum, urine and sweat were 97.4 % to 105.3 %, 96.4 % to103.4 %, and 95.1 % to 104.4 %, respectively. Current strategies based on PLNPs ratiometric sensors provide an effective method with high selectivity and accuracy for the detection of DA in clinical samples.

A novel ratiometric aptasensor based on SERS for accurate quantification of cardiac troponin I

Cardiac troponin I (cTnI) as a biomarker of acute myocardial infarction (AMI) is directly related to myocardial damage. Here, we proposed a ratiometric aptamer-sensor (aptasensor) for quantifying cTnI based on surface-enhanced Raman scattering (SERS), in which the uniform and high-density gold array was decorated with highly specific aptamer Tro4 to form the capture substrate for target cTnI. Firstly, gold array was labeled with 5, 5’-dithio bis-(2-nitrobenzoic acid) (DTNB) as one SERS signal to effectively realize signal self-calibration. In addition, aptamer Tro6 targeting cTnI and 4-mercaptobenzoic acid (4-MBA) as another SERS signal were anchored to porous gold (pAu) to form a pAu signal probe. In the presence of cTnI, the capture substrate and pAu signal probes could build a typical sandwich structure, thus the ratio of SERS peak intensity (I4-MBA/IDTNB) could be used to quantify cTnI. The ratiometric aptasensor showed a good linearity from 0.001 to 100 ng mL−1 and a low detection limit down to 0.27 pg mL−1. Own to dual signal output and the gold array substrate, the aptasensor exhibited good reproducibility, high sensitivity and accuracy. When the aptasensor was applied to the quantification of cTnI in human serum samples, the detection performance was comparable to that of enzyme-linked immunosorbent assay (ELISA), proving that the sensor has a good prospect in the diagnosis of AMI disease.

Regenerable ratiometric aptasensor based on electro-oxidation conducted host-guest dissociation for aflatoxin B1 detection in grains

Aflatoxin B1 (AFB1) leads to considerable pollution to grains and causes harmful effects on human health. Herein, we present a regenerable AFB1 aptasensor, based on electro-oxidation conducted host-guest dissociation for natural AFB1 detection. Molybdenum disulfide-Au nanobipyramids (MoS2-AuNBPs) serve as signal carriers and are equipped with indicative thionine (Thi) and semi-complementary chains to AFB1 aptamer (ssDNA). The electrode surface is modified with gold nanoparticles (AuNPs) and β-cyclodextrin (β-CD). Aptamer-ferrocene (Apt-Fc) can be recognized in cavity of β-CD through host-guest interaction. The signal probe, self-assembled with AFB1 Apt via hybridization, can be easily expelled by AFB1, bringing a ratiometric readout ΔIThi/IFc. Particularly, regeneration process is realized with synergy of AFB1-conducted Apt-ssDNA dissociation and electro-oxidation conducted host (β-CD)-guest (Fc) dissociation. Modifications and regeneration behaviors are morphologically and electrochemically validated. Regenerations are dependably performed in repetitive cycles (N = 7), which exhibit considerable performance in analyses. Electro-oxidation time is experimentally optimized (6 min). This method exhibits wide range from 1.0 pg/mL to 80.0 ng/mL, and limit of detection is 5.0 × 10−1 pg/mL, to target AFB1. During natural AFB1 determinations, proposed readouts are validated with certified HPLC and recovery test. Relative errors and recovery rates are calculated as 2.04–7.22% and 94.41–106.54%, respectively.

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.

Ratiometric electrochemical aptasensor based on split aptamer and Au-rGO for detection of aflatoxin M1

A novel ratiometric electrochemical aptasensor based on split aptamer and Au-reduced graphene oxide (Au-rGO) nanomaterials was proposed to detect aflatoxin M1 (AFM1). In this work, Au-rGO nanomaterials were coated on the electrode through the electrodeposition method to increase the aptamer enrichment. We split the aptamer of AFM1 into 2 sequences (S1 and S2), where S1 was immobilized on the electrode due to the Au-S bond, and S2 was tagged with methylene blue (MB) and acted as a response signal. A complementary strand to S1 (CS1) labeled with ferrocene (Fc) was introduced as another reporter. In the presence of AFM1, CS1 was released from the electrode surface due to the formation of the S1-AFM1-S2 complex, leading to a decrease in Fc and an increase in the MB signal. The developed ratiometric aptasensor exhibited a linear range of 0.03 μg L-1 to 2.00 μg L-1, with a detection limit of 0.015 μg L-1 for AFM1 detection. The ratiometric aptasensor also showed a linear relationship from 0.2 μg L-1 to 1.00 μg L-1, with a detection limit of 0.05 μg L-1 in natural milk after sample pretreatment, indicating the successful application of the developed ratiometric aptasensor. Our proposed strategy provides a new way to construct aptasensors with high sensitivity and selectivity.

An ultrasensitive ratiometric aptasensor based on the dual-potential electrochemiluminescence of Ru(bpy)32+ in a novel ternary system for detection of Patulin in fruit products.

To sensitively monitor trace-level of toxic patulin (PAT), an ultrasensitive PAT ratiometric aptasensor based on the dual-potential electrochemiluminescence (ECL) of Ru(bpy)32+ was first proposed. Noteworthily, Ru(bpy)32+-doped trimetallic nanocube (Ru@Tri) innovatively integrated the luminophore and cathode coreaction accelerator (CCA), which could generate strong cathodic ECL in the existence of low concentration of K2S2O8. Simultaneously, anthocyanin-derived carbon quantum dots (anth-CQDs) prepared from purple potato skins was first served as a green anodic coreactant. And SiO2-coated anth-CQDs (anth-CQDs@SiO2) exhibited excellent performance for enhancing anodic ECL of Ru@Tri. Based on this, a novel ternary ECL system was established. In the presence of PAT, the ECL intensity ratio of anode to cathode (IECL-A/IECL-C) was significantly increased, and a low detection limit of 0.05pgmL-1 was obtained. Moreover, when proposed method and high performance liquid chromatography (HPLC) were simultaneously applied to series of fruit products, the obtained results were completely consistent, reflecting its practicability.

A dual-mode ratiometric aptasensor for accurate detection of pathogenic bacteria based on recycling of DNAzyme activation

Pathogenic bacteria have a significant impact on food safety. Herein, an innovative dual-mode ratiometric aptasensor was constructed for ultrasensitive and accurate detection of Staphylococcus aureus (S. aureus) based on recycling of DNAzyme activation on gold nanoparticles-functionalized MXene nanomaterials (MXene@Au NPs). Electrochemiluminescent (ECL) emitter-labeled probe DNA (probe 2-Ru) containing the blocked DNAzyme was partly hybridized with aptamer and then captured by electrochemical (EC) indicator-labeled probe DNA (probe 1-MB) on electrode surface. When S. aureus presented, the conformation vibration of probe 2-Ru activated the blocked DNAzymes, leading to recycling cleavage of probe 1-MB and ECL tag close to electrode surface. Based on the reverse change tendencies of ECL and EC signals, aptasensor achieved S. aureus quantification from 5 to 108 CFU/mL. Moreover, the self-calibration characteristic of the aptasensor with dual-mode ratiometric readout ensured the reliable measurement of S. aureus in real samples. This work showed useful insight into sensing foodborne pathogenic bacteria.

A ratiometric aptasensor for simultaneous determination of two estrogens based on multicolor upconversion nanoparticles

Different environmental estrogens (EEs) may coexist in the same foodstuff, necessitating the establishment of a simultaneous detection method. Although there are numerous studies on the detection of EEs, few was reported on the determination of two or more estrogens at the same time except HPLC. Our efforts have been made to develop a ratiometric aptasensor based on multicolor lanthanide-doped upconversion luminescence nanoparticles (UCNPs) to realize simultaneous and sensitive detection of two EEs (bisphenol A and 17β-estradiol). Three types of core-shell UCNPs with excellent luminous properties and distinct emitting colors were successfully synthesized via doping with various lanthanide ions, two of which were conjugated with specific aptamers and act as energy donors, while another one served as a reference. Because of their excellent luminescence quenching performance, ultra-thin metallic MoS2 nanosheets were selected as the energy acceptor. Thus, a ratiometric luminescence aptasensor was established and successfully used for the simultaneous detection of two estrogens in tap water and milk samples, demonstrating its considerable practical application value.

Exogenous interference and autofluorescence-free ratiometric aptasensor for detection of OTA based on dual-colored persistent luminescence nanoparticles

Accurate and sensitive detection of ochratoxin A (OTA) is highly necessary due to its high carcinogenicity, teratogenicity and mutagenicity. Herein, we reported an exogenous interference and autofluorescence-free ratiometric aptasensor based on dual-colored persistent luminescent nanoparticles for precise detection of OTA. Green-emitting ZnGeO:Mn bonded with OTA aptamer and BHQ1-modified complementary base was acted as detection and specific recognition probe (ZGM@BHQ1). Quaternary ammonium modified ZnGaGeO:Cr with red emission was employed as reference probe and further bonded to ZGM@BHQ1 through electrostatic interaction to construct the ratiometric aptasensor. The developed ratiometric aptasensor was free from real-time excitation, external interference and autofluorescence and gave low detection limit of 3.4 pg mL−1, wide linearity in the range of 0.01–50 ng mL−1 and high precision of 3.1 % (11 replicate determinations, at 1 ng mL−1 level). The applicability of the aptasensor was successfully demonstrated by analyzing OTA in in grain samples with recoveries of 97.6 %-105.2 %.

Docking-aided rational tailoring of a fluorescence- and affinity-enhancing aptamer for a label-free ratiometric malachite green point-of-care aptasensor

Although nucleic acid aptasensors are increasingly applied in the detection of environmentally hazardous biomolecules, several formidable challenges remain with this technique because of their vulnerability, high cost and suboptimal sensitivity. Here, a docking-aided rational tailoring (DART) strategy was established at three levels and in two dimensions for the refinement of malachite green (MG) DNA aptamers. Guided by in silico molecular docking, coarse and fine tailoring were conducted at three levels each, to significantly enhance fluorescence activation intensity and binding affinity in two dimensions. Empowered by the results of the rational tailoring, a mechanistic view of the MG DNA aptamer-target interaction was thoroughly analyzed via four types of interactions. To meet the demand for point-of-care testing (POCT), a label-free and ratiometric fluorescent aptasensor was developed leveraging the tailored MG aptamer, based on the binding site competition-equilibrium effect via the introduction of a reference dye. This sensitive, specific, low-cost and rapid aptasensor subsequently demonstrated outstanding detection performance, achieving an ideal signal response range of 5 nmol·L−1 - 6 μmol·L−1 and a low limit of detection (LOD) of 1.49 nmol·L−1. The DART strategy and systematic exploration of the MG DNA luminescent aptamers herein will provide a valuable reference in the field of aptamer tailoring, biosensing and bioimaging. The proposed label-free ratiometric aptasensor also provides a highly generalizable strategy for hazardous biomolecular detection.

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.

Ratiometric fluorescent aptasensor for convenient detection of ochratoxin A in beer and orange juice.

Based on the principle of fluorescence resonance energy transfer (FRET), a simple ratiometric fluorescent aptasensor for convenient detection of ochratoxin A (OTA), a Group IIB carcinogen secreted by some fungi, was established. Initially, the anti-OTA aptamer with a quadruplex structure was flanked with FAM and BHQ1, and its partially complementary DNA (cDNA) was tagged with Cy3. In the absence of OTA, this aptamer hybridized with the cDNA strand forming a DNA duplex structure, in which BHQ1 was adjacent to Cy3 and distant from FAM. Due to the FRET principle, the fluorescence intensity emitted by Cy3 (FCy3) was quenched by BHQ1, and the fluorescence intensity emitted by FAM (FFAM) recovered. In the presence of OTA, the prepared aptamer preferred to bind with OTA instead of cDNA, forming an aptamer-OTA complex structure in which BHQ1 was adjacent to FAM and distant from Cy3. As a result, FFAM was quenched and FCy3 was restored. OTA can be accurately detected via the determination of the FCy3/FFAM ratio value. Under optimal conditions, this ratiometric fluorescent aptasensor offers excellent OTA detection in the range of 0.6 nmol L-1-5 μmol L-1, with a limit of detection (LOD) of 0.3 nmol L-1. This ratiometric aptasensor showed the advantages of easy operation, accuracy and sensitive analysis. Good specificity of this aptasensor was demonstrated. This ratiometric aptasensor could be used for the detection of OTA in real samples, e.g. beer and orange juice, showing its promising application potential.

Rational design of a ratiometric fluorescent aptasensor for patulin in traditional Chinese medicine through the studies of the interaction mechanism between its DNA aptamer and the target molecule.

Patulin (PAT) is a kind of mycotoxin which must be monitored for the sake of quality and safety in traditional Chinese medicine (TCM) owing to its harm to human health. On this account, a rationally designed ratiometric fluorescent aptasensor was developed based on the studies of the interaction mechanism between PAT and its aptamer (PAT-APT). First, CD spectroscopy, molecular docking, and molecular dynamic simulation were applied to investigate the details on how PAT-APT binds with its target molecule. The results indicated that the structure of PAT-APT changed to a certain extent and was stabilized after binding with PAT. C-11, C-37 and C-38 were the key sites for the recognition and interaction between PAT-APT and its target. Second, based on these results, a ratiometric aptasensor was designed using fluorescence resonance energy transfer (FRET) and synchronous fluorescence spectroscopy. A complementary sequence (cDNA) to the aptamer with an appropriate length and hybridization position was obtained through rational design and optimization. Both PAT-APT and cDNA were labeled using a pair of fluorophores, which could generate FRET when the two single-stranded oligonucleotides hybridized. The accurate detection of PAT could be realized according to the change ratio of the fluorescence intensity at the corresponding wavelengths of the two fluorophores before and after the assay. The aptasensor achieved an ultralow limit of detection of 0.16 nM, perfect selectivity, and satisfactory practicability in complex TCM samples. To our knowledge, this is the first aptasensor for PAT designed through the interaction mechanism between its aptamer and the target molecule. Moreover, the assay for PAT is cost-effective, does not need complicated pretreatment and only takes less than an hour. In summary, this study makes a contribution to the safety control of TCM and provides a thinking mode from mechanism to rational design to conquer the problem of sensitive aptasensing of one component in a complex system.

Competitive Ratiometric Aptasensing with Core-Internal Standard-Shell Structure Based on Surface-Enhanced Raman Scattering

Reproducibility and stability are important indicators for the evaluation of quantitative sensing methods based on surface-enhanced Raman scattering (SERS) technology. Developing a SERS substrate with self-calibration capabilities is vital for effectively quantifying targets. In this work, a competitive ratiometric SERS aptasensor was developed. 4-Aminothiophenol as an internal standard (IS) was embedded in the substrate followed by gradually loading with the aptamer and methylene blue functionalizing of the complementary sequences of the aptamer (MB-cDNA). Recognition and binding of the target to the aptamer resulted in the shedding of MB-cDNA after magnetic separation reducing the SERS signal of MB, allowing for the ratiometric determination of the target based on the constant intensity from the IS. For the selective detection of okadaic acid (OA), a good negative correlation was achieved between the SERS ratiometric intensity and OA concentration in the range of 0.5-100 ng/mL. The magnetic separation strategy effectively simplifies the production steps of the aptasensor, and the ratiometric strategy effectively improved the reproducibility and stability of the OA sensing. This ratiometric aptasensor has been successfully employed to detect OA in food and environmental samples and is expected to be extended to detect other targets.

Dual-quenching effects of methylene blue on the luminophore and co-reactant: Application for electrochemiluminescent-electrochemical ratiometric zearalenone detection

Methylene blue (MB) is a common multifunctional indicator, which can be applied as a quencher for electrochemiluminescence (ECL) analysis as well as a classical redox probe. Although it is relatively prevalent for MB to study the mechanism with Ru-based luminophores in ECL systems, there are few studies on the effects between MB and co-reactants. In this work, we proposed the first investigation of MB on the luminophore and co-reactant of the self-enhanced ECL composites (nitrogen-doped graphene quantum dots on Ru(bpy)32+-doped silica nanoparticles, NGQDs-Ru@SiO2), respectively. The relatively narrow ECL spectrum of luminophore (Ru@SiO2) and the suitable ultraviolet–visible absorption spectrum of MB led to the ECL resonance energy transfer between them, meanwhile the appropriate energy levels among them facilitated the electron transfer, resulting in a decreased ECL signal (quench mode I). Additionally, the co-reactant (NGQDs) was prone to π-π conjugation with MB due to its abundant π-electrons, which reduced the concentration of NGQDs’ intermediates and triggered a weakened ECL signal (quench mode II). Therefore, the dual-quenching effects are ingeniously integrated and designed in one ECL-electrochemical (ECL-EC) ratiometric aptasensor for zearalenone detection, for demonstrating its efficacy in enhancing the sensitivity, which is 4.8-fold higher than Ru@SiO2 alone. This innovative ratiometric aptasensor achieved a relatively wide linear range from 1.0 × 10−15 to 5.0 × 10−8 g mL−1, and obtained a low detection limit of 8.5 × 10−16 g mL−1. Our proposed dual-quenching interactions between MB and NGQDs-Ru@SiO2 will open a new prospective for ECL-EC ratiometric aptasensor, which further broaden the application in sensitive and precise analysis of mycotoxins.

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.

Ultrasensitive dual-signal electrochemical ratiometric aptasensor based on Co-MOFs with intrinsic self-calibration property for Mucin 1

The concentration of tumor biomarker Mucin 1 (MUC 1) is highly related with many diseases, which can be employed for the early diagnosis of cancer. In this paper, an electrochemical ratiometric aptasensor with intrinsic self-calibration property for the detection of MUC 1 is presented. In this paper, Co-MOFs themselves were employed as signal substances. This strategy was fabricated by using gold nanoparticles@black phosphorus (BP) as the substrate on the electrode, followed by modification of DNA nanotetrahedrons (DTN) via Au–S bond. The terminal of DTN contains MUC 1 aptamer. In the presence of MUC 1, the signal of DNA-labeled Co-MOFs can be detected. The current signal of Co-MOFs increased and that of thionine (as reference) was unchanged upon the addition of MUC 1. Thus, an intrinsic self-calibration aptasensor was achieved. In order to simplify the modification procedure, the electrolyte solution thionine was employed as an inner reference probe. Moreover, coupling of the hybridization chain reaction (HCR) with these MOFs signal tags presents an enzyme-free method for signal amplification, endowing the proposed ratiometric biosensor detection with high reproducibility and high sensitivity. The current ratio (IIR/ISP) remained stable over 30 individual measurements performed on ten different working electrodes. Even ten repeated scans performed on a single electrode exhibited a constant current ratio. The electrochemical ratiometric aptasensor is highly sensitivity for MUC 1 with the detection limit of 1.34 fM. Our proposed ratiometric sensor has great potential for the detection of cancer-related biomarkers.

Ratiometric fluorescent detection of pesticide based on split aptamer and magnetic separation

Pesticide residues have seriously threatened human health and affected the sustainable development of the environment. It is pivotal to establish an efficient detection platform for pesticide assays. A novel ratiometric fluorescent aptasensor for sensitive determination of pesticide has been successfully established. Carbon dots (CDs) are used as the reference emitter, while the T-rich single-stranded DNA templated copper nanoparticles (CuNPs) provide the increasing signal. More precisely, CDs/magnetic beads (MBs) combined with split aptamer are used in this detection platform to present a label-free and enzyme-free isocarbophos (ICP) detection with high sensitivity and specificity. In the presence of ICP, a sandwich-typed complex is formed which attaches a T-rich DNA. After magnetic separation and addition of Cu2+/ascorbic acid (AA), the CuNPs are generated and the strong red luminescent appears. Without ICP, just blue emission from CDs exits. The developed ratiometric aptasensor offers an excellent ICP detection in the linear range of 3–200 nM with a detection limit of 0.81 nM. It provides new ways for the assay of pesticides in agricultural products or environmental samples.

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 0.1-10000pgmL-1 with a detection limit of 0.012pgmL-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.