Electrochemical Biosensor For Ultrasensitive Detection Research Articles

The construction of COFs functionalized CRISPR electrochemical sensor for ultrasensitive detection of bacteria by hyper-branched rolling circle amplification

Rapid, sensitive, and low-cost foodborne pathogens detection is crucial for dealing with foodborne diseases caused by bacteria. In this work, we designed a new CRISPR/Cas9-mediated electrochemical biosensor for ultrasensitive detection of Escherichia coli (E. coli) based on multifunctional covalent organic framework-based nanocomposite (GOx-AuNPs-COF-H2) and hyperbranched rolling circle amplification (HB-RCA) reaction. When specific DNA targets were present in the system, the targets could be recognized and cleaved by CRISPR-Cas9 system, which triggered strand displacement reaction (SDR) and HB-RCA reaction. By introducing GOx-AuNPs-COF-H2 nanocomposite, H2 probe could hybridize with RCA products to bind to the electrode surface. After added glucose solution, GOx could catalyze it in the detection system and thus offered significant current signals for bacteria detection. This approach showed high analytical performance for assay of E. coli with the detection limit (LOD) of 10 CFU/mL. Furthermore, real samples were detected by this approach, demonstrating its promising potential for foodborne pathogens detection in food monitoring and clinical diagnosis.

A simple and reliable interenzyme distance regulation strategy based on a DNA quadrangular prism scaffold for ultrasensitive ochratoxin A detection

Developing sensitive and accurate Ochratoxin A (OTA) detection methods is essential for food safety. Herein, a simple and reliable strategy for regulating interenzyme distance based on a rigid DNA quadrangular prism as a scaffold was proposed to establish a new electrochemical biosensor for ultrasensitive detection of OTA. The interenzyme distances were precisely adjusted by changing the sequences of the hybridized portions of hairpins SH1 and SH2 to the DNA quadrangular prism, avoiding the complexity and instability of the previous DNA scaffold-based enzyme spacing adjustment strategies. The electrochemical biosensor constructed at the optimal interenzyme distance (10.4 nm) achieved sensitive detection of OTA in a dynamic concentration range from 10 fg/mL to 250 ng/mL with a detection limit of 3.1 fg/mL. In addition, the biosensor was applied to quantify OTA in real samples, exhibiting great application potential in food safety.

A novel electrochemical biosensor based on dual signal amplification of CMK-3@AuNPs and ATRP for DR1 detection

Down-regulator of transcription 1 (DR1) is a crucial biomarker closely related to Hashimoto's Thyroiditis (HT). In this study, a highly selective electrochemical biosensor for ultrasensitive detection of DR1 was prepared by combining nanomaterials CMK-3 @AuNPs and atom transfer radical polymerization (ATRP) signal amplification strategy for the first time. Based on the specific recognition of antigen and antibody, the sensor has strong selectivity. CMK-3 @AuNPs can not only increase the conductivity of the electrode, but also provide more active sites for biological molecules, so as to effectively amplify the output signal. The FMMA monomers were polymerized on the electrode surface through ATRP reaction, which made the electrochemical signal increased significantly. Under optimal conditions, the fabricated electrochemical biosensor showed a wide detection range of 5 × 10-4 ng/mL ∼ 5 × 102 ng/mL with a detection limit as low as 0.159 pg/mL. In addition, the electrochemical biosensor has good stability and anti-interference ability. Therefore, the sensor has broad prospects in the detection of biomarkers.

A novel cascade catalytic electrochemical biosensor based on bifunctional Mn3O4@AuNPs with self-supplied O2 property and glucose oxidase-like activity for ultrasensitive detection of microRNA

Herein, a bifunctional nanomaterial Mn3O4@AuNPs with self-supplied oxygen (O2) property and glucose oxidase-like activity was exploited to construct a novel cascade catalytic electrochemical biosensor for ultrasensitive detection of microRNA-499 (miRNA-499). Impressively, compared with the traditional AuNPs nanozyme with single glucose oxidase-like activity, the Mn3O4@AuNPs could not only act as a glucose oxidative mimetic enzyme to catalyze the oxidation of glucose to gluconic acid and the reduction of dissolved O2 to low concentration of hydrogen peroxide (H2O2) to obtain a electrochemical signal in the presence of hemin/G-quadruplex DNAzyme with peroxidase-like activity, but also electrocatalyze water splitting to obtain abundant dissolved O2 to further assist in the production of high concentration of H2O2 for a significantly improved electrochemical response. Meanwhile, with the aid of the target-induced dual Mg2+-assisted DNAzyme cleavage recycling amplification for the increasement of target miRNA-499 conversion efficiency, the proposed electrochemical biosensor achieved the ultrasensitive detection of miRNA-499 with a detection limit as low as 6.2 aM. As a result, this strategy might provide a potential method to upgrade the catalytic efficiency of mimic enzyme cascade reaction with eventual applications in bioanalysis and disease diagnosis.

Rolling Circle Amplification/G‐Quadruplex‐Based Dual‐Signal Ratiometric Electrochemical Aptasensor for Ultrasensitive Detection of Pathogenic Bacteria

AbstractAccurate and efficient detection of pathogenic bacteria plays a crucial role in the diagnosis of infectious diseases. However, rapid and highly sensitive detection of specific bacteria in clinical samples remains challenging. In this study, we developed a ratiometric dual‐signal electrochemical biosensor for ultrasensitive detection of pathogenic bacteria, based on an aptamer recognition‐induced rolling circle amplification (RCA)/G‐quadruplex strategy. On the surface of a gold electrode chip, we used an aptamer sequence (P1) conjugated to ferrocene (Fc) to capture the target bacterium. This capture released a different, previously bound sequence (P2) sequence that initiated the RCA/G‐quadruplex cascade, which, in the presence of potassium ions, was able to bind the electrochemical indicator methylene blue (MB). We integrated the signals from the loss of Fc and the appearance of MB (IMB/IFC ratio) to quantify the target bacteria concentration. This biosensor showed excellent detection performance and specificity, with a detection limit of 10 CFU/mL. Notably, it showed great diagnostic potential for clinical infectious diseases caused by pathogenic bacteria in precise/personalized medicine applications.

A bacterial cellulose-based and low-cost electrochemical biosensor for ultrasensitive detection of SARS-CoV-2

COVID-19 has led to over 6.8 million deaths worldwide and continues to affect millions of people, primarily in low-income countries and communities with low vaccination coverage. Low-cost and rapid response technologies that enable accurate, frequent testing of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants are crucial for outbreak prevention and infectious disease control. Here we produce and characterize cellulose fibers naturally generated by the bacterium Gluconacetobacter hansenii as an alternative biodegradable substrate for manufacturing an eco-friendly diagnostic test for COVID-19. Using this green technology, we describe a novel and label-free potentiometric diagnostic test that can detect SARS-CoV-2 within 10 min and costs US$3.50 per unit. The test has bacterial cellulose (BC) as its substrate and a carbon-based electrode modified with graphene oxide and the human angiotensin-converting enzyme-2 (ACE2) as its receptor. Our device accurately and precisely detects emerging SARS-CoV-2 variants and demonstrates exceptional sensitivity, specificity, and accuracy for tested clinical nasopharyngeal/oropharyngeal (NP/OP) samples.

Self-accelerated DNA walker mediated electrochemical biosensor for rapid and ultrasensitive detection of microRNA

Herein, we developed a novel three-dimensional (3D) self-accelerated DNA walker (SADW) which progressively expedite walking rate by unlocking the more walking arm continuously in walker process to construct electrochemical biosensor for ultrasensitive detection of microRNA. Particularly, we skillfully introduced a target analogue sequence in the double-loop hairpin, which could be released in the walking process of SADW, then rapidly activating more silenced walking strands to achieve the continuous self-acceleration, resulting in the expedited reaction rate. Surprisingly, the average reaction rate of SADW was quite higher than that of traditional 3D self-circulating DNA walkers (DW) under pretty low target miRNA concentration, which is ascribed to the outstanding acceleration process of the SADW, readily conquering the major predicaments of DW in detecting target with traces concentration: slow reaction rate and low sensitivity. This way, the elaborated SADW is favorably applied in the ultrasensitive and rapid detection of miRNA-21 in tumor cancer cell lysates with a detection limit down to 5.81 aM which was far from lower than the detection limit of DW. This approach develops the novel generation of widespread strategy for the applications in clinic diagnose, biosensing assay, and DNA nanobiotechnology.

An electrochemical biosensor based on a highly loaded 3D DNA nanostructure for ultrasensitive detection of Pb2+

Herein, a novel three-dimensional (3D) DNA nanostructure with highly loaded capacity of methylene blue (MB) as signal molecule was constructed to fabricate an electrochemical biosensor for ultrasensitive detection of Pb2+. First, a small amount of target Pb2+ could be converted into numerous mimic targets D1, S1, D2 and S1′ with the assistant of Pb2+-dependent DNAzyme cleavage cycling, which could trigger MB labeled hairpin DNA H3, H4, H5 and H6 to undergo hybridization chain reaction (HCR) for forming layer A and layer B, respectively. Then, the proposed 3D DNA nanostructure was constructed by layer-by-layer assembly of the prepared layer A and layer B labeled with abundant MB for endowing the 3D DNA structure with excellent carrying capacity of MB and generating a remarkable electrochemical signal. Impressively, when the number of the assembly layers reached four, the optimal electrochemical signal value was obtained for achieving the ultrasensitive detection of Pb2+ with a low detection limit of 2.61 pM. Consequently, the proposed strategy provided a promising approach to design a novel 3D DNA structure with high loading capacity of signal molecule to construct high-performance sensor platform for ultrasensitive detection of biomarkers in diseases diagnosis and environmental monitoring.

A novel CRISPR/Cas14a-based electrochemical biosensor for ultrasensitive detection of Burkholderia pseudomallei with PtPd@PCN-224 nanoenzymes for signal amplification

In this work, PtPd nanoparticles functionalized porphyrin metal-organic framework nanoenzymes (PtPd@PCN-224 nanoenzymes) are exploited as signal amplification tags to fabricate a rapid and ultrasensitive sensitive CRISPR/Cas14a-based electrochemical biosensor for Burkholderia pseudomallei (B. pseudomallei) specific DNA sequences detection. The prepared PtPd@PCN-224 nanoenzymes not only catalyze the reduction peak current of H2O2 to obtain a strong electrochemical signal output, but also provide massive active sites for the assembly of nucleic acids by Zr–O–P bonds. Besides, the designed target-activated CRISPR/Cas14a is able to recognize the target DNA sequences and further trigger the trans-cleavage of ssDNA for signal amplification. Benefiting from the target-activated CRISPR/Cas14a and PtPd@PCN-224 nanoenzymes, the developed electrochemical biosensor for B. pseudomallei DNA detection exhibits high sensitivity with detection of limit down to 12.8 aM and excellent specificity for distinguishing non-targeted bacteria. Moreover, the CRISPR/Cas14a-based electrochemical detection platform can also apply for other pathogenic bacteria diagnostic by well-designing sgRNA for target sequence recognition, possessing high flexibility and versatility in clinical diagnosis.

Ultrasensitive electrochemical biosensor for microRNA-377 detection based on MXene-Au nanocomposite and G-quadruplex nano-amplification strategy

MicroRNAs (miRNAs) are considered as potential biomarkers for early diagnosis and prognostic assessment of diabetic nephropathy. In this work, an electrochemical biosensor for ultrasensitive detection of miRNA-377 was constructed based on MXene-Au nanocomposites and G-quadruplex nano-amplification strategy. As a promising nanocarrier, taking advantage of leveraging synergistic effects between Au nanoparticles (AuNPs) and MXene nanosheet, MXene-Au nanocomposites exhibited excellent electronic conductivity and provided massive active sites for DNA capture probe immobilization by Au-S bonds. AuNPs modified with Guanine-rich sequence DNA detection probes were designed as signal amplification nano-labels. Specifically, by inducing the transition of Guanine-rich detection probes to G-quadruplex, the strong affinity interaction between methylene blue and G-quadruplex could not only reflect trace concentration information of miRNA-377, but also lead to the further enhancement of electrochemical signal (2.7-fold). As a result, this newly designed biosensor exhibited superior sensing performance with a wide linear range from 10 aM to 100 pM and the limit of detection was as low as 1.35 aM. Compared with biosensors based on other nanocomposites and reported miRNA-377 biosensors, the proposed sensing platform did not require any thermal cycling or reverse transcription process, meeting the miRNA sensing requirements of convenient, sensitive, specific and stable. Furthermore, the as-constructed biosensor also displayed good selectivity, which was applied to accurate detection of miRNA-377 in human serum samples with satisfactory sensitivity, suggesting the biosensor system has promising applications in biological researches and early clinical diagnosis for diabetic nephropathy.

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.

A Novel Crispr/Cas14a-Based Electrochemical Biosensor for Ultrasensitive Detection of Burkholderia Pseudomallei with Ptpd@Pcn-224 Nanoenzymes for Signal Amplification

A Novel Crispr/Cas14a-Based Electrochemical Biosensor for Ultrasensitive Detection of Burkholderia Pseudomallei with Ptpd@Pcn-224 Nanoenzymes for Signal Amplification

Dual 3D DNA Nanomachine-Mediated Catalytic Hairpin Assembly for Ultrasensitive Detection of MicroRNA.

Herein, we designed a dual 3D DNA nanomachine (DDNM)-mediated catalytic hairpin assembly (DDNM-CHA) to construct an electrochemical biosensor for ultrasensitive detection of miRNA, which possesses quite a faster reaction rate and much higher amplification efficiency than those of traditional catalytic hairpin assembly (CHA). Impressively, since the DDNM skillfully increases the local concentration of reactants and decreases the steric hindrance of substrates simultaneously, the DDNM-CHA could be endowed with higher collision efficiency and more effective reaction compared with traditional CHA, resulting in a hyper conversion efficiency up to 2.78 × 107 only in 25 min. This way, the developed DDNM-CHA could easily conquer the main predicaments: long reaction time and low efficiency. As a proof of the concept, we adopt the gold nanoparticles (AuNPs) and the magnetic nanoparticle (Fe3O4) as the kernel of DNM-A and DNM-B, respectively, and harness the magnetic electrode to directly adsorb the products H1-H2/Fe3O4 for constructing an immobilization-free biosensor for high-speed and ultrasensitive detection of miRNA with a detection limit of 0.14 fM. As a result, the DDNM-CHA we developed carves out a new insight to design a functional DNA nanomachine and evolve the analysis method for practical amplification in the sensing area and promotes the deeper exploration of the nucleic acid signal amplification strategy and DNA nanobiotechnology.

Co-catalytic Fc/HGQs/Fe3O4 nanocomposite mediated enzyme-free electrochemical biosensor for ultrasensitive detection of MicroRNA.

Herein, a novel co-catalytic ferrocene/hemin/G-quadruplexes/Fe3O4 nanoparticles (Fc/HGQs/Fe3O4) nanocomposite was synthesized to significantly magnify the electrochemical signal of ferrocene (Fc) using the synergistic catalysis of hemin/G-quadruplexes (HGQs) and Fe3O4 nanoparticles as hydrogen peroxide enzyme mimics for the construction of ultrasensitive electrochemical biosensors. The fabricated electrochemical biosensor can achieve ultrasensitive detection of miRNA-155 ranging from 0.1 fM to 1 nM, as well as a limit of detection of 74.8 aM. This strategy provides a new route to exploring efficient signal labels for signal amplification and provides an impetus to find novel methods for the construction of biosensors for biological detection and the early clinic diagnosis of diseases.

A double-tetrahedral DNA framework based electrochemical biosensor for ultrasensitive detection and release of circulating tumor cells.

Detecting circulating tumor cells (CTCs) in patients' blood is essential for early diagnosis, precise treatment and prognosis of cancer. Yet due to CTCs being extremely rare in the peripheral blood of patients, it is still a challenge to detect CTCs with high sensitivity and high selectivity. Here, we developed a double-tetrahedral DNA framework (DTDF) based electrochemical biosensor system (E-CTC sensor system) for ultrasensitive detection and release of CTCs. In this work, an upright tetrahedral DNA framework (UTDF) was used as a rigid scaffold to modify a screen-printed gold electrode (SPGE), and an inverted tetrahedral DNA framework (ITDF) provided three vertex chains to multivalently bind with aptamers. Meanwhile, a streptavidin tagged horseradish peroxidase homopolymer (SA-polyHRP) was linked to biotin-modified aptamers to significantly amplify the signal. Moreover, the captured CTCs could be effectively released via benzonase nuclease with little cell damage. Our E-CTC sensor system achieved a linear range from 1 to 105 MCF-7 cells with an ultralow detection limit of 1 cell. The release efficiency reached 88.1%-97.6% and the viability of the released cells reached up to 98%. We also detected the MCF-7 cells in mimic whole blood samples, suggesting that the E-CTC sensor system shows promise for use in clinical research.

Dual signal amplification by polysaccharide and eATRP for ultrasensitive detection of CYFRA 21–1 DNA

Cytokeratin fragment antigen 21–1 (CYFRA 21–1) DNA is a crucial biomarker closely associated with non-small cell lung cancer. Here, we fabricated a novel electrochemical biosensor for ultrasensitive detection of CYFRA 21–1 DNA via polysaccharide and electrochemically mediated atom transfer radical polymerization (eATRP) dual signal amplification. Specifically, thiolated peptide nucleic acid (PNA) probes at 5′-terminals are immobilized on the gold electrode surface for specific recognition of CYFRA 21–1 DNA (tDNA). After hybridization, hyaluronic acid (HA) is linked to the hybridized PNA/DNA duplexes via the recognized carboxylate-Zr4+-phosphate chemistry. Then multiple initiators of the polymerization reaction are introduced via esterification reaction. Lastly, large numbers of electro-active monomers are successfully grafted from the initiation sites of functionalized HA by eATRP reaction, significantly amplifying the electrochemical signal. Under optimal conditions, the constructed sensor can detect as low as 9.04 aM tDNA. Further, this proposed biosensor can also be applied to the direct detection of double-stranded DNA (dsDNA), obtaining 0.12 fM as the detection limit. Besides, this strategy shows high selectivity for mismatched bases and excellent applicability for CYFRA 21–1 DNA detection in the serum samples. Given its high sensitivity, selectivity, ease of operation, low cost and environmental friendliness, this biosensor has considerable potential in early diagnosis and biomedical application.

One DNA circle capture probe with multiple target recognition domains for simultaneous electrochemical detection of miRNA-21 and miRNA-155

In this work, a novel DNA circle capture probe with multiple target recognition domains was designed to develop an electrochemical biosensor for ultrasensitive detection of microRNA-21 (miRNA-21) and miRNA-155 simultaneously. The DNA circle capture probe was anchored at the top of the tetrahedron DNA nanostructure (TDN) to simultaneously recognize miRNA-21 and miRNA-155 through multiple target recognition domains under the assistance of Helper strands, which could trigger mimetic proximity ligation assay (mPLA) for capturing the beacons ferrocene (Fc)-A1 and methylene blue (MB)-A2 to achieve multiple miRNAs detection. In this way, the local reaction concentration could be enhanced and avoid the interference of various capture probes compared with the traditional multiplexed electrochemical biosensor with the use of different capture probes, resulting in the significantly improvement of detection sensitivity. As a result, this proposed biosensor showed wide linearity ranging from 0.1 fM to 10 nM with detection limits of miRNA-21 and miRNA-155 as 18.9 aM and 39.6 aM respectively, which also could be applied in the simultaneously detection of miRNA-21 and miRNA-155 from cancer cell lysates. The present strategy paved a new path in the design of capture probes for achieving more efficient and sensitive multiple biomarkers detections and possessed the potential applications in clinical diagnostic of diseases.

Target-driven rolling walker based electrochemical biosensor for ultrasensitive detection of circulating tumor DNA using doxorubicin@tetrahedron-Au tags

In this study, a multiple-legged and highly integrated DNA rolling walker based electrochemical biosensor was developed for ultrasensitive ctDNA analysis through rolling circle amplification (RCA) with doxorubicin@tetrahedron-Au (DOX@TDN-Au) as electrochemical indicator. Upon target-driven RCA, the multiple-legged walker could move along with the predesigned track by strand displacement reactions, resulting in numbers of legs binding irreversibly to iStep probes. The binding of massive legs to iStep probes could effectively impede DOX@TDN-Au tags binding on the surface of sensor and then reached a “signal off” state. Benefiting from the highly amplified efficiency of rolling walker machine and DOX@TDN-Au tags, the established biosensor performed high sensitivity for target detection with a low limit of detection down to 0.29 fM. Moreover, the target ctDNA could hybridize with the ring and capture probe simultaneously, greatly enhancing the specificity of the developed biosensing method. Thus, this biosensing method is a promising tool for detection of ctDNA in the field of clinical diagnostic and tumor progression assessment.

Electrocatalytic Efficiency Regulation between Target-Induced HRP-Mimicking DNAzyme and GOx with Low Background for Ultrasensitive Detection of Thrombin

Herein, an efficient target-activated enzyme cascade electrocatalysis with low background signal was employed to establish electrochemical biosensor for ultrasensitive detection of thrombin via regulating electrocatalytic efficiency between target-induced hemin/G-quadruplex horseradish peroxidase-mimicking DNAzyme (HRP-mimicking DNAzyme) and glucose oxidase (GOx).Impressively, only when the target thrombin was introduced, the HRP-mimicking DNAzyme acting simultaneously as electrochemical signal probe would be formed to activate high-efficiency enzyme cascade electrocatalysis for reducing background signal significantly, which could overcome the defect of inevitable high background signal during the detection of target in the traditional cascade electrocatalysis of two existing bioenzymes. In addition, the detection sensitivity could be further improved by regulating the side length of rigid DNA tetrahedron (TDN) scaffold anchored HRP-mimicking DNAzyme and GOx at adjacent vertices for high enzyme cascade electrocatalytic efficiency. Consequently, the proposed biosensor demonstrated a low detection limit down to 0.3 fM for target thrombin, which provided a promising method for ultrasensitive monitoring of biomolecules in sensing analysis and disease diagnosis.

Ultrasensitive electrochemical microRNA-21 biosensor coupling with carboxylate-reduced graphene oxide-based signal-enhancing and duplex-specific nuclease-assisted target recycling

An electrochemical biosensor for ultrasensitive detection of microRNA-21 (miRNA-21) coupling with carboxylate-reduced graphene oxide (COOH-rGO)-based signal-enhancing and duplex-specific nuclease (DSN)-assisted target recycling. Capture DNA (cDNA) was self-assembled on the surface of gold electrode (GE) and hybridized with the target miRNA-21 to form cDNA/miRNA-21 hybrids. COOH-rGO was further aggregated on cDNA/miRNA-21 hybrids through π − π stacking interaction, leading to a accumulation of electroactive methylene blue (MB) molecules and a significant differential pulse voltammetry (DPV) response of MB with peak potential of −0.28 V (vs. Ag/AgCl). At the present of DSN, DSN digested the cDNA/miRNA-21 hybrids and released miRNA-21 to hybridize with another cDNA, resulting in a significant decrease of the length of cDNA sequence. The amount of MB on the electrode surface decreased dramatically, following with the decrease of the DPV signal of MB. Under optimal experimental conditions, this approach displayed an ultrahigh sensitivity (with a 0.01 fM detection limit) and excellent selectivity among similar miRNAs sequences even with single-base difference. This biosensor further exhibited excellent applicability for target miRNA-21 in human serum samples with satisfactory results.