Ratiometric Electrochemical Biosensor Research Articles

Ratiometric electrochemical biosensor based on hybridization chain reaction signal amplification for sensitive microRNA-155 detection.

In this work, a sensitive ratiometric electrochemical biosensor for microRNA-155 (miRNA-155) detection is reported based on a hybridization chain reaction amplifying the electrochemical signal. The biosensor was fabricated using Au NPs as a modified material to assemble capture DNA labeled with ferrocene (Fc) molecules, and a DNA probe labeled with methylene blue (MB) was employed for the signal probe. In the presence of target miRNA-155, it can be dual hybridized with capture and signal probe, especially with signal probe to continuously produce long concatemers containing lots of MB molecules. The electrochemical signal of Fc was used for the internal signal, and the signal from MB was used as an indicator signal. As the concentration of miRNA-155 was altered, the internal reference signal of Fc remained constant, and only the indicator signal changed in a sensitive way. The change in the ratio (IMB/IFc) between the indicator signal of MB and internal reference signal of Fc can be used to monitor the concentration of miRNA-155. Under optimal conditions, the prepared ratiometric biosensor could detect miRNA-155 within a wide linear range from 100 fM to 100 nM with low detection limit of 33 fM (at S/N = 3). Moreover, the biosensor was evaluated with human serum samples, and satisfactory recoveries were obtained, indicating that the ratiometric biosensor can be applied to clinical sample analysis.

Ratiometric electrochemical biosensor based on Cu(II) modified covalent organic framework for the ultra-sensitive and specific detection of glutathione

As a vital biological polypeptide molecule, glutathione (GSH) is involved in the regulation of various life activities. Accurate and sensitive detection of GSH in human body is of great significance for the preliminary diagnosis of related diseases. In this research, Cu(II) modified TADH COF, which has been prepared by Cu2+ post-synthesis modification of the COF obtained through a condensation reaction of 1,3,5-(4-aminophenyl) benzene (TAPB) and 2,5-dihydroxy-1,4-benzenedicarboxaldehyde (DHTP), and methylene blue (MB) molecules are combined to construct a ratiometric electrochemical sensor for achieving specific and ultra-sensitive detection of GSH. As a stable and active covalent organic framework material, TADH COF possesses the special O(–OH) and N(CN) binding sites, which can load and coordinate with Cu2+ effectively. The obtained Cu(II)-TADH COF serves as a sensitive electrode material for sensing GSH due to “extrusion effect” caused CuCl solid-state electrochemical signal degeneration by the specific binding of Cu and GSH while MB provides a stable electrical signal in the electrochemical sensing process as an internal reference for eliminating errors and making the detection results more accurate. The constructed electrochemical sensing platform Cu(II)-TADH COF/MB/GCE exhibited an ultrahigh sensitivity with a limit of detection (LOD) of 0.0093 nM (S/N = 3), wide linear range of 0.05 nM–40 μM and high accuracy. For the detection of GSH in real samples including human blood, serum and urine samples, the sensor also showed excellent sensing performance. This is the first research on ratiometric electrochemical sensing platform for the ultra-sensitive detection of glutathione based on covalent organic frameworks, which provides an original and novel strategy for the electrochemical detection of GSH with COF materials.

An anti-fouling surface molecularly imprinted ratiometric electrochemical biosensor for SARS-CoV-2 spike protein

An anti-fouling surface molecularly imprinted (SMI) ratiometric electrochemical biosensor was fabricated for the detection of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) spike protein (S protein). Polypyrrole (PPy) was electrodeposited on the electrode surface by using ferrous phthalocyanine (PcFe) and sodium alginate (SA) as the dopants, which acted as the molecularly imprinted polymers (MIPs). The SMI electrochemical biosensor could be used for the detection of SARS-CoV-2 S protein based on the ratiometric signals of the internal reference (PcFe) and the external probe ([Fe(CN)6]3−/4−). Under optimized conditions, the as-fabricated SMI ratiometric biosensor showed a wide linear range of 100 fg mL−1 ∼ 100 ng mL−1 with a low limit of detection (LOD) of 30.1 fg mL−1. Nonspecific adsorption of interfering substances onto the SMI biosensor surface could be significantly reduced due to the outstanding anti-fouling ability of SA, as revealed by fluorescence microscopy. Finally, the reliability of the SMI ratiometric electrochemical biosensor was demonstrated by the detection of SARS-CoV-2 S protein in clinical serum samples. The findings of this work open a new avenue in clinical diagnosis of COVID-19.

A ratiometric electrochemical biosensor based on ARGET ATRP for detection of HER2 gene

Human epidermal growth factor receptor 2 (HER2), a common proto-oncogene, is overexpressed in a subset of breast cancer patients. It is essential to track HER2 expression for early breast cancer diagnosis. Herein, a ratiometric electrochemical biosensor for detection of HER2 based on activators generated by electron transfer for atom transfer radical polymerisation (AGET ATRP) and hairpin DNA was developed. Specifically, hairpin DNA was first self-assembled on the gold electrode by Au–S bond. Upon capturing HER2, the stem-loop structure of hairpin DNA was unfolded, the signal value of methylene blue (MB) decreased as it moved away from the electrode surface. cDNA was linked with HER2 by complementary base pairing to introduce amino group. Then, the initiator 2-bromo-2-methylpropionic acid (BMP) were connected to the amino group on the cDNA to activate ARGET ATRP. The detection performance of biosensors for HER2 was explored by the ratio signal between two signal molecules. Under optimal conditions, this ratiometric electrochemical biosensor shows good selectivity and stability with a wide detection range of 1–1 × 106 pM and a detection limit of 78.47 fM. Furthermore, the biosensor exhibits satisfactory anti-interference ability due to the hairpin DNA and dual signal system, and has promising application prospects in the detection of other DNA disease markers.

Development of a highly sensitive aptamer-based electrochemical sensor for detecting saxitoxin based on K3Fe(CN)6 regulated silver nanoparticles

BackgroundSaxitoxin (STX) is the most toxic marine toxin, which can pose several adverse effects on human health. High sensitivity, fast response, and low-cost detection of STX contamination are of significance to reducing the fishery and seafood industries' loss. Among the various types of biosensors, the electrochemical biosensors have been extensively studied in the detection of STX, but the electrode surface modification material is easy to fall off, resulting in unstable electrochemical signals and poor reproducibility. It is imperative to have a ratiometric electrochemical biosensor for STX. ResultsIn this study, we developed a novel aptamer-based electrochemical sensor (AECs) for the sensitive detection of STX based on a K3Fe(CN)6 regulated silver nanoparticles (Ag NPs) modified with aptamer. The AECs was constructed by immobilizing aptamer on Ag NPs surfaces. Under optimized conditions, the AECs showed a linear response towards STX in the range from 0.04 to 0.15 μM with the regression equation of Y = −8.0 + 233.7 X (R2 = 0.9956). The limit of detection (LOD) was calculated to be 1 nM (based on 3 N/S), which is significantly lower than the regulatory limits for STX in seafood. Moreover, the AECs showed excellent sensitivity, reproducibility and stability, as well as the detection in samples with acceptable recovery ranged from 71.2 % to 93.8 %, demonstrating its broad application prospects in detection of STX in seafood samples. SignificanceThis work proposed an AECs to achieve sensitive detection of STX. A reaction system of K3Fe(CN)6 etched Ag NPs was introduced and used as the signal source to avoid the instability of the electrochemical signal, which can produce a ratiometric electrochemical signal output mode, improving the stability and sensitivity of electrochemical detection of STX.

Ratiometric electrochemical biosensor based on lateral movement of multi-pedal DNA tetrahedron machine on biomimetic interface

In this work, a lateral moving multi-pedal DNA tetrahedron machine (MTM) is designed and coupled with dual-signal output system to construct a biomimetic electrochemical ratiometric strategy for ultrasensitive target DNA analysis. The tetrahedral structure provided rigid support for the pedal, ensuring efficient replacement of the rail chain modified with ferrocene. By conjugating cholesterol molecules to one vertex of MTM, it is decorated on a lipid bilayer. This molecular architecture confers lateral movement of MTM on an electrode surface while prevents its detachment from the system. The methylene blue tagged hairpin probe provides constant power to support MTM swim on lipid bilayer. Compared with the conventional motion mode, the lateral moving mechanism has the fastest reaction rate and the highest signal-to-noise ratio. Additionally, the dual-signal reporting system further improves the accuracy of target detection on the basis of ensuring motion efficiency. The work improved movement efficiency and shortened time fragment. A linear relationship between the ratio value of two reporters and target DNA concentration was observed from 0.5 fM to 50 pM with a detection limit of 28 aM. The lateral motion mode of DNA machine coalescing with ratiometric system made this sensing platform ultrasensitive and accurate, which holds new avenue of early diagnosis.

Dual DNA recycling amplifications coupled with Au NPs@ZIF-MOF accelerator for enhanced electrochemical ratiometric sensing of pathogenic bacteria

Sensitive and accurate quantification of pathogenic bacteria is vastly significant to the related food safety. Herein, a sensitive ratiometric electrochemical biosensor was developed for the detection of Staphylococcus aureus (S. aureus) based on dual DNA recycling amplifications and Au NPs@ZIF-MOF accelerator. Gold nanoparticles-loaded Zeolitic imidazolate metal-organic framework (Au NPs@ZIF-MOF) as electrode substrate possessed a large specific surface area for nucleic acid adsorption, and as an accelerator promoted the transfer of electrons. The strong recognition of aptamer to target S. aureus could initiate the padlock probe-based exponential rolling circle amplification (P-ERCA, as the first DNA recycling amplification), generating large numbers of trigger DNA strands. The released trigger DNA further activated the catalytic hairpin assembly (CHA, as the second DNA recycling amplification) on electrode surface. Consequently, P-ERCA and CHA continuously brought about one target to many signal transduction, leading to an exponential amplification. To achieve the accuracy of detection, the signal ratio of methylene blue (MB) and ferrocene (Fc) (IMB/IFc) was applied for intrinsic self-calibrating. Taking advantages of dual DNA recycling amplifications and Au NPs@ZIF-MOF, the proposed sensing system displayed high sensitivity for S. aureus quantification with a linear range of 5–108 CFU/mL, and the limit of detection was 1 CFU/mL. Moreover, this system represented excellent reproducibility, selectivity, and practicability for S. aureus analysis in foods.

CuS quantum dots activated DNAzyme for ratiometric electrochemical detection of telomerase activity.

Telomerase activity detection has attracted much attention concerning its importance for early cancer diagnosis. Here, we established a ratiometric electrochemical biosensor for telomerase detection based on CuS quantum dots (CuS QDs) dependent DNAzyme-regulated dual signals. The telomerase substrate probe was used as the linker to combine the DNA fabricated magnetic beads and CuS QDs. In this way, telomerase extended the substrate probe with repeated sequence to from hairpin structure, releasing CuS QDs as an input to DNAzyme modified electrode. DNAzyme was cleaved with high current of ferrocene (Fc) and low current of methylene blue (MB). On the basis of the obtained ratiometric signals, telomerase activity detection was achieved in the range of 1.0×10-12-1.0×10-6 IU/L, with the limit of detection down to 2.75×10-14 IU/L. Moreover, telomerase activity from HeLa extracts was also tested to verify the clinical application.

An electrochemical ratiometric biosensor for the detection of dopamine based on an MXene-Au nanocomposite.

Compared with single signal detection, a ratiometric biosensor could offer more accurate and reliable results. Here, a ratiometric electrochemical biosensor for the sensitive and accurate detection of dopamine was developed based on the strong adsorption ability of MXene-Au toward methylene blue, an inner reference element. This ratiometric sensing strategy opened up a new avenue for the development of a ratiometric platform.

Pathogen Identification: Ultrasensitive Nucleic Acid Detection via a Dynamic DNA Nanosystem-Integrated Ratiometric Electrochemical Sensing Strategy.

Sensitively determining trace nucleic acid is of great significance for pathogen identification. Herein, a dynamic DNA nanosystem-integrated ratiometric electrochemical biosensor was proposed to determine human immunodeficiency virus-associated DNA fragment (HIV-DNA) with high sensitivity and selectivity. The dynamic DNA nanosystem was composed of a target recycling unit and a multipedal DNA walker unit. Both of them could be driven by a toehold-mediated strand displacement reaction, enabling an enzyme-free and isothermal amplification strategy for nucleic acid determination. The target recycling unit could selectively recognize HIV-DNA and activate the multipedal DNA walker unit to roll on the electrode surface, which would lead to bidirectional signal variation for ratiometric readout with cascade signal amplification. Benefiting from the synergistic effect of the dynamic DNA nanosystem and the ratiometric output mode, the ultrasensitive detection of HIV-DNA was achieved in a wide linear range of 6 orders of magnitude with a limit of detection of 36.71 aM. The actual usability of the proposed sensor was also verified in complex biological samples with acceptable performance. This dynamic DNA nanosystem-integrated ratiometric sensing strategy might be promising in the development of reliable point-of-care diagnostic devices for highly sensitive and selective pathogen identification.

An ultrasensitive and specific ratiometric electrochemical biosensor based on SRCA-CRISPR/Cas12a system for detection of Salmonella in food

Sensitive and specific detection of Salmonella is highly desirable to guarantee food safety and prevent food-borne disease outbreaks. Herein, we proposed a ratiometric electrochemical biosensor based on the combination of SRCA and CRISPR/Cas12a system for ultrasensitive and specific detection of Salmonella. Signal amplification is accomplished by reasonably integrating rapid SRCA amplification and the trans-cleavage capabilities of Cas12a. Accordingly, the non-specific amplification was eliminated. Compared with single signal-based electrochemical approaches, the ratiometric electrochemical measurements offer self-calibration to reduce the internal/external disturbances, thereby achieving precise analytical performance and high reliability. At this strategy, based on the ratio of the current signal of ferrocene (Fc) and methylene blue (MB) (IFc/IMB), the biosensor displayed a linear range from 5.8 fg/μL to 5.8 ng/μL and detected as low as 2.08 fg/μL of Salmonella in pure cultures. For the detection of the actual samples, this biosensor was 100% sensitivity, 97.8% specificity and 98% accuracy in comparison with the international standard. And the detection results of this biosensor were consistent with real-time fluorescent quantitative PCR (RT-qPCR), which further verified the accuracy and practical application value of the biosensor. Overall, this strategy provides a valuable platform for highly specific and ultrasensitive screening of Salmonella in food.

Construction of a sensitive ratiometric electrochemical sensing platform for DNA methylation detection based on the design of multistep DNA amplification circuits

Methylated DNA is a promising epigenetic biomarker for diseases diagnosis, therapy monitoring, and prognosis. Here, we developed a sensitive ratiometric electrochemical biosensor for the detection of methylated DNA based on the design of multistep DNA amplification circuits. The methylated target DNA firstly initiates the DNAzyme-assisted amplification circuit and produces a number of single-stranded triggers that catalyze the subsequent double-step catalytic hairpin assembly circuits to release numerous four-way DNA junctions. The four-way DNA junctions displace the methylene blue (MB)-modified signal probes for intercalating doxorubicin (DOX) molecules on the sensing interface, resulting in a significantly increased value of iDOX/iMB to achieve sensitive monitoring of methylated DNA. Taking advantage of the significant signal amplification of multistep DNA amplification circuits, the proposed method exhibited high sensitivity for target analysis with a detection limit of 4 aM and a dynamic linear range of 10 aM to 20 pM. Moreover, the proof-of-concept application of the sensing platform was investigated, suggesting that the system is promising for potential applications in early cancer diagnosis and the fundamental research of epigenetics.

Metal-Organic Framework UiO-66-Mediated Dual-Signal Ratiometric Electrochemical Sensor for microRNA Detection with DNA Walker Amplification.

Electrochemical nanotags with strong signal input are necessary for a ratiometric electrochemical sensor to overcome the drawbacks of inaccurate detection results. In this paper, the metal-organic framework (MOF) UiO-66 was utilized as an electrochemical signal tag. A stable and strong current response at +0.9 V can be detected in neutral conditions. MicroRNA (miRNA) was employed as the model analyte. Herein, an enzyme-free DNA-walker-based ultrasensitive ratiometric electrochemical biosensor in combination with Zr MOF (UiO-66) signal tags to detect miRNA was demonstrated. In the presence of miRNA, the autonomous walker movement can be initiated by miRNA, leading to the release of biotin-modified fragments. Thus, streptavidin-labeled UiO-66 nanomaterials were not bound to the electrode, generating a low signal response of UiO-66 at +0.9 V. However, the current signal of electrolyte solution as reference at +0.2 V was increased due to the enhancement of electrode conductivity. This ratiometic sensor demonstrated high sensitivity, selectivity, and reproducibility. It can eliminate the disturbance of environmental factors and basic electrode characteristics, providing more accurate signals. A limit of detection (LOD) of 0.17 fM was achieved. Moreover, the method was also used to detect miRNA-21 spiked in real serum samples.

Recent development and application of ratiometric electrochemical biosensor

• Different categories of ratiometric electrochemical biosensors are described. • The applications of ratiometric electrochemical biosensors are discussed. • The main challenges and prospects in this field are proposed. As one kind of sensitive analysis devices, electrochemical biosensors (ECBSs) convert biological activities into quantifiable electrical signals depending on biological recognitions. Due to their simple operation, low background signal, high sensitivity and stable performance, ECBSs have attracted widespread attentions. However, majority of ECBSs adopt a single-signal output method, which may inevitably be affected by environment conditions and operating procedures, thus leading to inaccurate results. In order to overcome these issues, ratiometric electrochemical biosensors (RECBSs) have been explored. In RECBSs, internal reference substances are introduced to provide a built-in correction, which endow the biosensors with superior sensitivity and accuracy. This article reviews different types of RECBSs, as well as their latest applications in the detection of proteins, nucleic acids, small biomolecules and ions. In addition, major challenges in this field are outlined. Some suggestions for their developments in the future are also highlighted.

Biosensor Based on Covalent Organic Framework Immobilized Acetylcholinesterase for Ratiometric Detection of Carbaryl.

A ratiometric electrochemical biosensor based on a covalent organic framework (COFThi-TFPB) loaded with acetylcholinesterase (AChE) was developed. First, an electroactive COFThi-TFPB with a two-dimensional sheet structure, positive charge and a pair of inert redox peaks was synthesized via a dehydration condensation reaction between positively charged thionine (Thi) and 1,3,5-triformylphenylbenzene (TFPB). The immobilization of AChE on the positively charged electrode surface was beneficial for maintaining its bioactivity and achieving the best catalytic effect; therefore, the positively charged COFThi-TFPB was an appropriate support material for AChE. Furthermore, the COFThi-TFPB provided a stable internal reference signal for the constructed AChE inhibition-based electrochemical biosensor to eliminate various effects which were unrelated to the detection of carbaryl. The sensor had a linear range of 2.2–60 μM with a detection limit of 0.22 μM, and exhibited satisfactory reproducibility, stability and anti-interference ability for the detection of carbaryl. This work offers a possibility for the application of COF-based materials in the detection of low-level pesticide residues.

Programmable Analytical Feature of Ratiometric Electrochemical Biosensor by Alternating the Binding Site of Ferrocene to DNA Duplex for the Detection of Aflatoxin B1†

Comprehensive SummaryRatiometric biosensor is among the promising strategies for the advanced analysis; its applicability in multiple scenarios, however, is seriously limited for the fixed analytical feature. Here, we reported ratiometric electrochemical biosensors with programmable analytical feature by alternating the binding site of ferrocene (Fc) to DNA duplex for the detection of aflatoxin B1 (AFB1). DNA duplex was synthesized with AFB1 aptamer and its complementary DNA (cDNA) with Fc molecular tagged on aptamer or cDNA. For analysis, aptamer‐AFB1 complex stripped from the electrode, and free cDNA triggered hybridization chain reaction to produce double‐stranded DNA for methylene blue (MB) absorption. In this way, redox currents of Fc (IFc) and absorbed MB (IMB) at the electrode were collected to produce the ratiometric signal of IMB/IFc as a yardstick for AFB1. When Fc was tagged on aptamer, IFc decreased with higher concentration of AFB1 (cAFB1), the resultant biosensor allowed detection with an On/Off (IMB/IFc) mode; when Fc was tagged on cDNA, IFc kept constant as cAFB1 varying, offering an On/Constant (IMB/IFc) mode for the detection. Consequently, biosensor with On/Off mode showed a linear range of 0.1—100 pg/mL with a limit of detection (LOD) of 0.0650 pg/mL, while biosensor with On/Constant mode offered a wider linear rang of 1—10000 pg/mL but a relatively poor LOD of 0.330 pg/mL. Our work revealed the vital importance of binding site of probes to DNA, and this principle can endow ratiometric biosensor with programmable analytical feature (e.g., linear range, LOD), allowing detections in various scenarios.

An ultrafast ratiometric electrochemical biosensor based on potential-assisted hybridization for nucleic acids detection

The development of rapid nucleic acid detection methods, which are promising the diagnostic standard of the infectious disease, could expand more options for disease traceability and controllability. Nucleic acid hybridization-based biosensing techniques still encounter limitations in meeting the requirements for rapid detection. Therefore, we proposed a potential-assisted ratiometric electrochemical biosensor for rapid and accurate target DNA detection. This dual-signal analysis system actuated an enzyme-free detection process. The application of an external electric field accelerated molecular dynamics, resulting in highly efficient collision chances. This hybridization process was thus improved from hours to minutes compared with passive hybridization approach. The biosensor not only had a high sensitivity with the detection limit of 12 fM, but also features a robust capability in identifying single-base mismatch. Moreover, the biosensor realized sensitive detection of target DNA in complex biological environments. Overall, this sensing strategy exhibits a promising potential for application in point-of-care testing.

Catalytic Hairpin Assembly-Driven Ratiometric Dual-Signal Electrochemical Biosensor for Ultrasensitive Detection of MicroRNA Based on the Ratios of Fe-MOFs and MB-GA-UiO-66-NH2.

In this work, a novel ratio electrochemical biosensing platform based on catalytic hairpin assembly target recovery to trigger dual-signal output was developed for ultrasensitive detection of microRNA (miRNA). To achieve the ratiometric dual-signal strategy, methylene blue (MB), an electrochemical indicator, was ingeniously loaded into the pores of graphene aerogel (GA) and metal-organic framework (MOF) composites with high porosity and large specific surface area, and another electrochemical indicator Fe-MOFs with distinct separation of redox potential was selected as a signal probe. Concretely, with the presence of the target miRNA, the CHA process was initiated and the signal probe was introduced to the electrode surface, producing abundant double-stranded H1-H2@Fe-MOFs-NH2. Then, the measurement and analysis of the prepared ratiometric electrochemical biosensor by differential pulse voltammetry (DPV) showed that the introduction of the target miRNA led to an increase in the oxidation peak signal of Fe-MOFs (+0.8 V) and a decrease in the oxidation peak signal of MB (-0.23 V). Therefore, the peak current ratio of IFe-MOFs/IMB could be employed to accurately reflect the actual concentration of miRNA. Under optimal conditions, the detection limit of the proposed biosensor was down to 50 aM. It was worth noting that the proposed biosensor exhibited excellent detection performance in a complex serum environment and tumor cell lysates, showing great potential in biosensing and clinical diagnosis.

Magnetic antifouling material based ratiometric electrochemical biosensor for the accurate detection of CEA in clinical serum

A novel ratio electrochemical biosensor based on multi-functional nanocomposite was developed. Fe3O4 was synthesized in situ on carboxyl functionalized 2D nanomaterial MXene, and then covalently bonded with [Ru(NH3)6]3+ to obtain nanocomposites MXC-Fe3O4-Ru. Fe3O4 and [Ru(NH3)6]3+ can neutralize the electronegativity of the MXene to make the nanocomposites electrically neutral. Combine with the good hydrophilicity and conductivity of MXene, the nanocomposites can be utilized to construct antifouling electrochemical biosensors without modifying with specific antifouling materials. Moreover, Fe3O4 can endow the nanocomposites with magnetism, and [Ru(NH3)6]3+ is used as an internal standard molecule. The strong magnetic MXC-Fe3O4-Ru can be easily separated and firmly modified on the magnetic gold electrode (MGE). DNA double-stranded (dsDNA) containing an ferrocene (Fc)-modified carcinoembryonic antigen (CEA) aptamer can be specifically captured to the surface of the electrode by amido bond. In the presence of CEA, CEA binds to the aptamer and leaves the electrode surface, the electrochemical signal of Fc decreases, while the electrochemical signal of [Ru(NH3)6]3+ is fixed on the electrode surface remains basically unchanged. The ratio of the electrochemical signals of Fc and [Ru(NH3)6]3+ is proportional to the CEA concentration. The linear range of the sensor is 1 pg/mL to 1 μg/mL with a detection limit of 0.62 pg/mL. With the excellent antifouling performance, good conductivity of the nanocomposite, and the application of the ratiometric strategy, the biosensor can achieve high selectivity, accuracy, and sensitivity for the detection of targets even in complex samples, such as FBS and clinical serum.

A Novel Ratiometric Electrochemical Biosensor Using Only One Signal Tag for Highly Reliable and Ultrasensitive Detection of miRNA-21.

Herein, a novel ratiometric electrochemical biosensor with methylene blue (MB) as the only one signal tag was proposed for highly reliable and ultrasensitive detection of microRNA-21 (miRNA-21) under the assistance of an intelligent target-induced dual signal amplification (T-DSA). First, a small amount of target miRNA-21 could produce abundant mimic targets DNA S1 and Zn2+ through target-induced recycle and acid dissolution, respectively. Then, S1 triggered rolling circle amplification (RCA) to generate functional DNA nanospheres (DSP) encoded by DNAzyme and substrate sequence for loading numerous signal tag MB with a remarkable electrochemical signal (signal on), and the Zn2+ cofactor mediated the nonviolent DNAzyme-catalyzed cleavage of DSP to sharply release MB with obviously reduced electrochemical responses (signal off). Impressively, our strategy could controllably load and release the only signal tag MB through the well-designed DSP to effectively avoid the false positive responses caused by the non-ideal upright state of DNA probes connected to electrodes in traditional distance-dependent signal adjustment ratiometric strategies with two different signal tags. Meanwhile, with the aid of innovative T-DSA recycle and RCA-produced functional DSP, the detection sensitivity of this sensing platform was significantly improved. As a result, the proposed biosensor successfully realized highly reliable and ultrasensitive detection of miRNA-21 with a detection limit down to 26.7 aM, which shows exceptional promise in biological analysis and medical diagnosis.