Catalytic Hairpin Assembly Research Articles

Ultrasound-enhanced catalytic hairpin assembly capable of ultrasensitive microRNA biosensing for the early screening of Alzheimer's disease

Catalytic hairpin assembly (CHA) is a promising enzyme-free, isothermal signal amplification strategy, but the relatively time-consuming strand replacement limits its application scenarios. Here, we developed an ultrasound-enhanced catalytic hairpin assembly (UECHA) biosensing platform for early screening of Alzheimer's disease by introducing a portable acoustic-drive platform with functionalized microspheres for effective biomarkers enrichment and fluorescence enhancement. By constructing a gradient ultrasonic field in a microcavity, the platform concentrates the functionalized microspheres in a central position, accompanied by an enhanced fluorescence signal with a specific release. In addition, the programmable frequency modulation can also modify the acoustic potential well and effectively promote non-equilibrium chemical reactions such as CHA (25 min). Compared with the conventional catalytic hairpin assembly (CHA), UECHA allows for direct and quantitative measurement of AD miRNAs down to 3.55 × 10−15 M in 1 μL samples. This visual analysis of ultra-trace biomarkers based on acoustic enrichment and promotion provides a new perspective for the rapid and highly sensitive clinical detection of Alzheimer's disease.

Base-Stacking-Driven Catalytic Hairpin Assembly: A Nucleic Acid Amplification Reaction Using Electrode Interface as a "Booster" for SARS-CoV-2 Point-of-Care Testing.

Electrochemical DNA (E-DNA) biosensors based on interface-mediated hybridization reactions are promising for point-of-care testing (POCT). However, the low efficiency of target recycle amplification and the steric hindrance at the electrode interface limit their sensing performance. Herein, we propose a base-stacking-driven catalytic hairpin assembly (BDCHA), a nucleic acid amplification reaction strategy, for POCT. The introduction of the base-stacking effect in this strategy increases the thermodynamic stability of the product, thereby effectively improving the recycling efficiency. Also, it enables the interface-mediated hybridization to maintain stability with even fewer bases in the reaction-binding domain, hence minimizing DNA secondary structure formation or intertwining at the electrode surface and ameliorating the steric hindrance limitation. The introduced base-stacking effect makes the electrode serve as a "booster" by integrating the advantages of homogeneous and heterogeneous reactions, giving BDCHA an increased reaction rate of about 20-fold, compared to the conventional catalytic hairpin assembly. As a proof of concept, our BDCHA was applied in constructing a portable E-DNA biosensor for the detection of a SARS-CoV-2 N gene sequence fragment. A simple 30 min one-pot incubation is required, and the results can be readily read on a smartphone, making it portable and user-friendly for POCT.

A Cascade Signal Amplification Strategy for the Ultrasensitive Fluorescence Detection of Cu2+ via λ-Exonuclease-Assisted Target Recycling with Mismatched Catalytic Hairpin Assembly.

Herein, an ultrasensitive DNAzyme-based fluorescence biosensor for detecting Cu2+ was designed using the cascade signal amplification strategy, coupling λ-exonuclease-assisted target recycling and mismatched catalytic hairpin assembly (MCHA). In the designed detection system, the target, Cu2+, can activate the Cu2+-dependent DNAzyme to cause a cleavage reaction, releasing ssDNA (tDNA). Then, tDNA binds to hairpin DNA (H0) with an overhanging 5'-phosphorylated terminus to form dsDNA with a blunt 5'-phosphorylated terminus, which activates the dsDNA to be digested by λ-Exo and releases tDNA along with another ssDNA (iDNA). Subsequently, the iDNA initiates MCHA, which can restore the fluorescence of carboxyfluorescein (FAM) previously quenched by tetramethylrhodamine (TAMRA), resulting in a strong fluorescent signal. Furthermore, MCHA efficiently improves the signal-to-noise ratio of the detection system. More importantly, tDNA recycling can be achieved with the λ-Exo digestion reaction to release more iDNA, efficiently amplifying the fluorescent signal and further improving the sensitivity to Cu2+ with a detection limit of 60 fM. The practical application of the developed biosensor was also demonstrated by detecting Cu2+ in real samples, proving it to be an excellent analytical strategy for the ultrasensitive quantification of heavy metal ions in environmental water sources.

Integration of Isothermal Enzyme-Free Nucleic Acid Circuits for High-Performance Biosensing Applications.

The isothermal enzyme-free nucleic acid amplification method plays an indispensable role in biosensing by virtue of its simple, robust, and highly efficient properties without the assistance of temperature cycling or/and enzymatic biocatalysis. Up to now, enzyme-free nucleic acid amplification has been extensively utilized for biological assays and has achieved the highly sensitive detection of various biological targets, including DNAs, RNAs, small molecules, proteins, and even cells. In this Review, the mechanisms of entropy-driven reaction, hybridization chain reaction, catalytic hairpin assembly and DNAzyme are concisely described and their recent application as biosensors is comprehensively summarized. Furthermore, the current problems and the developments of these DNA circuits are also discussed.

Aggregation-Induced Enhanced Electrochemiluminescence from Tris(bipyridine)ruthenium(II) Derivative Nanosheets for the Ultrasensitive Detection of Human Telomerase RNA.

The traditional tris(bipyridine)ruthenium(II) complex suffers from the notorious aggregation-caused quenching effect, which greatly compromises its electrochemiluminescence (ECL) efficiency, thus hindering further applications in biosensing and clinical diagnosis. Here, the ultrathin tetraphenylethylene-active tris(bipyridine)ruthenium(II) derivative nanosheets (abbreviated as Ru-TPE NSs) are synthesized through a protein-assisted self-assembly strategy for ultrasensitive ECL detection of human telomerase RNA (hTR) for the first time. The synthesized Ru-TPE NSs exhibit the aggregation-induced enhanced ECL behavior and excellent water-dispersion. Surprisingly, up to a 106.5-fold increase in the ECL efficiency of Ru-TPE NSs is demonstrated compared with the dispersed molecules in an organic solution. The restriction of intramolecular motions is confirmed to be responsible for the significant ECL enhancement. Therefore, this proposed ECL biosensor shows high sensitivity and excellent selectivity for hTR based on Ru-TPE NSs as efficient ECL beacons and the catalytic hairpin assembly as signal amplification, whose detection limit is as low as 8.0fm, which is far superior to the previously reported works. Here, a promising analytical method is provided for early clinical diagnosis and a new type of efficient ECL emitters with great application prospects is represented.

Nucleic Acid‐to‐Small Molecule Converter through Amplified Hairpin DNA Circuits

AbstractMany microRNAs (miRNAs) are characteristically found in cancer cells, making miRNAs promising marker biomolecules for cancer diagnosis and therapeutics. However, it is challenging to use miRNA as a cancer signature because it is difficult to convert the nucleic acid sequence information into molecular functionality. To address this challenge, we realize nucleic acid‐to‐small molecule converters using hairpin DNA circuits. Harnessing a Staudinger reduction as a trigger for the conversion, we constructed hybridization chain reaction (HCR) and catalytic hairpin assembly (CHA) circuits that respond to oncogenic miR‐21. Fluorophore and dye molecules were released in response to miR‐21 through the HCR, providing fluorogenic and chromogenic readouts. Selective cytotoxicity in miR‐21‐abundant cells was realized by the CHA to release the anticancer drug SN‐38. This would be the first example of selective activation of a small‐molecule prodrug triggered by oncogenic miRNA in human living cells.

Nucleic Acid-to-Small Molecule Converter through Amplified Hairpin DNA Circuits.

Many microRNAs (miRNAs) are characteristically found in cancer cells, making miRNAs promising marker biomolecules for cancer diagnosis and therapeutics. However, it is challenging to use miRNA as a cancer signature because it is difficult to convert the nucleic acid sequence information into molecular functionality. To address this challenge, we realize nucleic acid-to-small molecule converters using hairpin DNA circuits. Harnessing a Staudinger reduction as a trigger for the conversion, we constructed hybridization chain reaction (HCR) and catalytic hairpin assembly (CHA) circuits that respond to oncogenic miR-21. Fluorophore and dye molecules were released in response to miR-21 through the HCR, providing fluorogenic and chromogenic readouts. Selective cytotoxicity in miR-21-abundant cells was realized by the CHA to release the anticancer drug SN-38. This would be the first example of selective activation of a small-molecule prodrug triggered by oncogenic miRNA in human living cells.

DNA Nanomachine-Driven Heterogeneous Quadratic Amplification for Sensitive and Programmable miRNA Profiling.

Inspired by the molecular crowding effect in biological systems, a novel heterogeneous quadratic amplification molecular circuit (HEQAC) was developed for sensitive bimodal miRNA profiling (HEQAC-BMP) by combining an MNAzyme-based DNA nanomachine with an entropy-driven catalytic hairpin assembly (E-CHA) autocatalytic circuit. Utilizing ferromagnetic nanomaterials as the substrate for DNA nanomachines, a biomimetic heterogeneous interface was established; thus, a localized molecular crowding system was created that can elevate the local reaction concentration and accelerate the molecular recognition process for a significant threshold signal. Simultaneously, the threshold signal undergoes further amplification by E-CHA and is transformed into a chemical signal, enabling a colorimetric-fluorescence bimodal signal readout. The HEQAC-BMP enables miRNA detection from 10 aM to 10 nM with detection limits of 3.7 aM (colorimetry) and 4.8 aM (fluorometry), respectively. Moreover, the design principle and strategy of HEQAC-BMP can be customized to address other critical viruses or diseases with life-threatening and socioeconomic impacts, enhancing healthcare outcomes for individuals.

Orderly Aggregated Catalytic Hairpin Assembly for Synchronous Ultrasensitive Detecting and High-Efficiency Co-Localization Imaging of Dual-miRNAs in Living Cells.

In this work, the orderly aggregated catalytic hairpin assembly (OA-CHA) was developed for synchronous ultrasensitive detection and high-efficiency colocalization imaging of dual-miRNAs by a carefully designed tetrahedral conjugated ladder DNA structure (TCLDS). Exactly, two diverse hairpin probes were fixed on tetrahedron conjugated DNA nanowires to form the TCLDS without fluorescence response, which triggered OA-CHA in the aid of output DNA 1 and output DNA 2 produced by targets miRNA-217 and miRNA-196a cycle to generate TCLDS with remarkable fluorescence response. Impressively, compared with the traditional CHA strategy, OA-CHA avoided the fluorescence group and quenching group from approaching again because of the spatial confinement effect to significantly enhance the fluorescence signal, resulting in the simultaneous ultrasensitive detection of dual-miRNAs with detection limits of 21 and 32 fM for miRNA-217 and miRNA-196a, respectively. Meanwhile, the TCLDS with lower diffusivity could achieve accurate localization imaging for reflecting the spatial distribution of dual-miRNAs in living cells. The strategy based on OA-CHA provided a flexible and programmable nucleic amplification method for the synchronous ultrasensitive detection and precise imaging of multiple biomarkers and had potential in disease diagnostics..

Proximity hybridization induced bipedal DNA walker for label-free electrochemical detection of apolipoprotein A4 based on DNA meditated Ag nanoparticles growth

Apolipoprotein A4 (Apo-A4) is considered as a prospective molecular biomarker for diagnosis of depression due to its neurosynaptic toxicity. Here, we propose a neighboring hybridization induced catalyzed hairpin assembly (CHA) driven bipedal DNA walker that mediates hybridization of Ag nanoparticles (Ag NPs) with DNA probes for highly sensitive electrochemical quantitative detection of Apo-A4. Driven by CHA, this bipedal DNA walker can spread all over the surface of the sensor, induce the HP1-HP2 double chain structure, make the surface of the sensor negatively charged, and adsorb a large number of Ag ions. After chemical reduction with hydroquinone, the Ag NPs formed provide signal tracers for electrochemical dissolution analysis of the target. The Ag NPs formed by chemical reduction of hydroquinone can provide signal traces for electrochemical stripping analysis of target thrombin. The linear range of this method is from 10 pg mL−1 to 1000 ng mL−1, and the detection limit is 5.1 pg mL−1. This enzyme-free and labeling detection method provides a new strategy for rapid clinical detection of Apo-A4 and accurate identification of depression.

DNAzyme-driven bipedal DNA walker and catalytic hairpin assembly multistage signal amplified electrochemical biosensor based on porous AuNPs@Zr-MOF for detection of Pb2+

As a highly toxic and refractory heavy metal contaminant, Pb2+ seriously endangers human health. The problems of low sensitivity and high cost of signal labeling widely exist in common electrochemical biosensors. Herein, a Pb2+ electrochemical biosensor was constructed using a DNAzyme-driven bipedal DNA Walker and catalytic hairpin assembly as the multistage signal amplification strategy. Compared with Zr-MOF, AuNPs@Zr-MOF has a larger porosity and specific surface area, which can effectively load MB to amplify the current signal. Pb2+ can trigger a dual signal amplification reaction to gradually accumulate the signal of methylene blue/gold nanoparticle @ zirconium-based metal organic frameworks (MB/AuNPs@Zr-MOF) on the electrode. The ingeniously designed sensing strategy realized the analysis of Pb2+ with a wide linear range from 0.05 to 1000 nmol/L and a lower limit of detection (LOD) of 4.65 pmol/L. In addition, the sensor has strong anti-interference ability and can accurately detect Pb2+ in various food samples.

A novel isothermal amplification strategy for rapid and sensitive detection of Matrix Metalloproteinase 2 using a bipedal DNA walker in anti-aging research

In the field of anti-aging research, a groundbreaking study introduces a novel isothermal amplification strategy for the rapid and sensitive detection of Matrix Metalloproteinase 2 (MMP-2) utilizing a bipedal DNA walker. MMP-2 plays a crucial role in skin aging due to its involvement in the degradation of collagen and elastin, key components of the extracellular matrix. The dysregulation of MMP-2 activity contributes to the breakdown of the extracellular matrix, leading to manifestations of skin aging such as wrinkles, sagging, and loss of elasticity. This research presents a novel method for detecting MMP-2 using a bipedal DNA walker. The method uses peptide nucleic acid (PNA) as a substrate for MMP-2, which can release a bandage strand that forms the bipedal walker “leg” with two DNA strands. The bipedal walker “leg” initiates the bipedal walker isothermal amplification strategy that involves entropy-driven DNA walking and catalytic hairpin assembly on a PEI@Ru(bpy)32+-Ti3C2 @AuNPs-modified electrode. The walker’s motion produces an electrochemiluminescence (ECL) signal that can be rapidly analyzed by a biosensor. The detection limit of this method is 80.6 fM, which is significantly lower than other methods. Overall, this study represents an important step towards the development of effective anti-aging research methods.

Research progress in the detection of common foodborne hazardous substances based on functional nucleic acids biosensors.

With the further improvement of food safety requirements, the development of fast, highly sensitive, and portable methods for the determination of foodborne hazardous substances has become a new trend in the food industry. In recent years, biosensors and platforms based on functional nucleic acids, along with a range of signal amplification devices and methods, have been established to enable rapid and sensitive determination of specific substances in samples, opening up a new avenue of analysis and detection. In this paper, functional nucleic acid types including aptamers, deoxyribozymes, and G-quadruplexes which are commonly used in the detection of food source pollutants are introduced. Signal amplification elements include quantum dots, noble metal nanoparticles, magnetic nanoparticles, DNA walkers, and DNA logic gates. Signal amplification technologies including nucleic acid isothermal amplification, hybridization chain reaction, catalytic hairpin assembly, biological barcodes, and microfluidic system are combined with functional nucleic acids sensors and applied to the detection of many foodborne hazardous substances, such as foodborne pathogens, mycotoxins, residual antibiotics, residual pesticides, industrial pollutants, heavy metals, and allergens. Finally, the potential opportunities and broad prospects of functional nucleic acids biosensors in the field of food analysis are discussed.

Enzyme-free nucleic acid and luminescent material cascade amplifications enable urinary dual miRNAs assay for non-invasive bladder cancer diagnosis

We presented a facile biosensing strategy for the detection of dual microRNAs (miRNA-21 and miRNA-126) in urine samples from bladder cancer (BC) patients. The fluorescent homogeneous detection of miRNAs was achieved by elaborate parallel catalytic hairpin assembly (CHA) integrated with material cascade amplifications. The continuous release of G-quadruplex (G4) sequences and Hg2+ was attributed to the specific recognition between the miRNAs and the corresponding hairpin oligonucleotides. Assisted by the streptavidin-(T-Hg2+-T-biotin)4 structures, more Hg2+ emission was obtained, and the spatial site block effect was used to order its distribution, significantly improving the selective recognition performance of cadmium telluride quantum dots (CdTe QDs) (1.4–2.9 times). Furthermore, the peroxidase-like activity of G4-hemin was used to catalyze the oxidation of dopamine (DA) to fluorescence-enhanced polydopamine (PDA). The generated PDA signal and the Hg2+-quenched CdTe QDs signal completed the synchronous detection to achieve a high-sensitivity analysis of the dual miRNAs. The limit of detection (LOD) of both miRNAs was as low as the fM level. Eventually, 56 clinical urine samples were quantified. The results showed satisfying specificity (100%) and sensitivity (92%) with a strong clinical agreement, including computed tomography (CT) and pathology findings, indicating a rapid non-invasive solution for early diagnosis of BC.

Simplified Rapid Enrichment of CTCs and Selective Recognition Prereduction Enable a Homogeneous ICP-MS Liquid Biopsy Strategy of Lung Cancer.

The effective enrichment and hypersensitivity analysis of circulating tumor cells (CTCs) in clinical whole blood samples are highly significant for clinical tumor liquid biopsy. In this study, we established an easy operation and affordable CTCs extraction technique while simultaneously performing the homogeneous inductively coupled plasma mass spectrometry (ICP-MS) determination of CTCs in lung cancer clinical samples based on selective recognition reactions and prereduction phenomena. Our strategy allowed for the pretreatment of whole blood samples in less than 45 min after step-by-step centrifugation, which only required lymphocyte separation solution and erythrocyte lysate. Furthermore, a three-stage signal amplification system consisting of catalytic hairpin assembly (CHA), selective recognition for C-Ag+-C structures and Ag+ of copper sulfide nanoparticles (CuS NPs), and prereduction of Hg2+ through ascorbic acid (AA) was constructed by using mucin 1 as the CTCs marker and the aptamer for identification probes. In optimal conditions, the detection limits of ICP-MS were as low as 0.3 ag/mL for mucin 1 and 0.25 cells/mL for A549 cells. This method analyzed CTCs in 58 clinical samples quantitatively, and the results were consistent with clinical CT images and pathological findings. The area under the curve (AUC) value of the receiver operating characteristic (ROC) curve was 0.957, which provided a specificity of 100% and a sensitivity of 91.5% for the assay. Therefore, the simplicity of the extraction method, the accessibility, and the high sensitivity of the assay method make the strategies attractive for clinical CTCs testing applications.

A sensitive and rapid electrochemical biosensor for sEV-miRNA detection based on domino-type localized catalytic hairpin assembly

Small extracellular–vesicule-associated microRNA (sEV-miRNA) is an important biomarker for cancer diagnosis. However, rapid and sensitive detection of low-abundance sEV-miRNA in clinical samples is challenging. Herein, a simple electrochemical biosensor that uses a DNA nanowire to localize catalytic hairpin assembly (CHA), also called domino-type localized catalytic hairpin assembly (DT-LCHA), has been proposed for sEV-miRNA1246 detection. The DT-LCHA offers triple amplification, (i). CHA system was localized in DNA nanowire, which shorten the distance between hairpin substrate, inducing the high collision efficiency of H1 and H2 and domino effect. Then, larger numbers of CHAs were triggered, capture probe bind DT-LCHA by exposed c sites. (ii) The DNA nanowire can load large number of electroactive substance RuHex as amplified electrochemical signal tags. (iii) multiple DT-LCHA was carried by the DNA nanowire, only one CHA was triggered, the DNA nanowire was trapped by the capture probe, which greatly improve the detection sensitivity, especially when the target concentration is extremely low. Owing to the triple signal amplification in this strategy, sEV-miRNA at a concentration of as low as 24.55 aM can be detected in 20 min with good specificity. The accuracy of the measurements was also confirmed using reverse transcription quantitative polymerase chain reaction. Furthermore, the platform showed good performance in discriminating healthy donors from patients with early gastric cancer (area under the curve [AUC]: 0.96) and was equally able to discriminate between benign gastric tumors and early cancers (AUC: 0.77). Thus, the platform has substantial potential in biosensing and clinical diagnosis.Graphical

Target aided self-assembly of DNA hyperbranched nanostructures for bacterial 16 S ribosomal DNA gene SERS detection

Quantity analysis of bacterial 16 S rDNA gene is still a challenge. Here we successfully achieved bacterial 16 S rDNA gene SERS analysis with target aided self-assembly of DNA hyperbranched nanostructures. Catalytic hairpin assembly (CHA) was first introduced for self-assembly of branched nanostructures, including target bacterial DNA served as trigger probe and three hairpin probes for further target activated cross opening. Rolling cycle amplification (RCA) was then introduced by using a circular DNA as template and single stranded branches of the formed branched nanostructures as primer, forming a concatemer containing multiple copies of tandem repeating segments with same sequences complementary to the circular templates. After that, thousands of Raman-Au nanoprobe would hybridize this long single stranded DNAs for DNA-AuNPs nanoaggretates assembly. By using SERS signal collection, target bacterial DNAs would be successfully analyzed with ultra-sensitivity as low as 15.0 fM. Furthermore, selectivity test for targets with mismatched DNA sequences and other bacterial 16 S rDNA genes show good results. This sensing method was extended to quantify target genes in human plasma samples, showing its excellent applicability in complex samples.

Rapid and portable bunyavirus SFTSV RNA testing utilizing catalytic hairpin assembly coupled with lateral flow immunoassay.

Severe fever with thrombocytopenia syndrome (SFTS) is a prevalent, life-threatening, emergent infectious disease. Currently, reverse transcription-polymerase chain reaction is the gold standard for diagnosing SFTS virus (SFTSV) infection, which requires sophisticated equipment and professional personnel that are frequently unavailable in most SFTS endemic rural areas. Here, we reported a simple, rapid nucleic acid amplification system that combined the catalytic hairpin assembly (CHA) with a lateral flow immunoassay (LFIA) strip-based detection method for SFTSV detection. The detection of SFTSV RNA could be realized by generation of H1-H2 hybrid duplexes labeled with biotin and digoxin, which subsequently added to the LFIA test strips containing streptavidin conjugated with Alexa Fluor 647 as well as anti-digoxin antibodies. Our CHA-based LFIA assay offered high amplification efficiency and specificity with a detection limit of 1 aM. Crucially, this method enabled stable detection of 500 copies/mL of SFTSV within 30 min using clinical serum samples. Therefore, our CHA-based LFIA approach provided a potential useful tool to facilitate early and precise diagnosis of SFTS patients in poorly resourced SFTS endemic areas.IMPORTANCESevere fever with thrombocytopenia syndrome (SFTS) is an emerging and potentially fatal infectious disease prevalent in China. Here we report a simple, rapid nucleic acid amplification system, the catalytic hairpin assembly (CHA) in conjunction with a lateral flow immunoassay (LFIA) strip-based detection method for SFTS virus detection, which demonstrated high amplification efficiency and specificity with limit of detection of 1 aM. Most importantly, we also validate our CHA-based LFIA assay using the clinical serum samples, which was fully compatible with reverse transcription-PCR results. Therefore, our strategy provides a potential useful tool to facilitate early and precise diagnosis of SFTS patients especially in poorly resourced SFTS endemic areas.

All-in-one nanoflare biosensor combined with catalyzed hairpin assembly amplification for in situ and sensitive exosomal miRNA detection and cancer classification

Exosomal miRNAs can reflect tumor progression and metastasis, and are effective biomarkers for cancer diagnosis. However, the accuracy of exosomal miRNA-based cancer diagnosis is limited by the low sensitivity and complicated RNA extraction of traditional approaches. Herein, a novel biosensor is developed for in situ, extraction-free, and highly sensitive analysis of exosomal miRNAs via nanoflare combined with catalyzed hairpin assembly (CHA) amplification. Without cumbersome and costly miRNA extraction or transfection agents, nanoflare can directly enter the exosomes to bind target miRNAs and generate a fluorescence signal that can be amplified by the CHA reaction to achieve the in situ and highly sensitive detection of exosomal miRNAs. Under the optimal conditions, the detection limit of 5 aM is obtained for three exosomal miRNAs, which is an order of magnitude lower than quantitative real time polymerase chain reaction (qRT-PCR). In combination with the linear discriminant analysis algorithm, five exosomes are distinguished with 100% accuracy. Importantly, five cancers including breast, lung, liver, cervical, and colon cancer from 64 patients are distinguished with 99% accuracy by testing exosomal miRNAs in clinical plasma. This simple, accurate, and sensitive biosensor holds the potential to be expanded into clinical non-invasive cancer diagnostic tests.

A dual-cycle amplification-based electrochemical platform for sensitive detection of tobramycin

BackgroundTobramycin (TOB), an essential aminoglycoside antibiotic in human life, poses potential threats due to its residues in the environment. The primary concern is the adverse impact of excessive TOB on human kidneys, hearing, and other organs, significantly affecting human health. Constructing a sensitive electrochemical platform for simple and rapid trace detection is crucial. Herein, to enhance the sensitivity of TOB detection in the environment and mitigate the risks associated with residual antibiotics, an ultrasensitive electrochemical aptasensor was developed. ResultsThe sensor employs a dual-cycle amplification strategy involving catalytic hairpin assembly (CHA) and exonuclease III (Exo III) for efficient signal amplification. Simultaneously, the electrode performance was optimized by incorporating gold nanowires (AuNWs) onto the surface of reduced graphene oxide (PDA-rGO). Specifically, in the presence of TOB, which binds to the aptamer (Apt), dsDNA dissociates, releasing cDNA to open hairpin 1 (HP1) and initiate the CHA cycle with the participation of hairpin 2 (HP2). Exo III shears HP1 in the HP1/HP2 complex, freeing HP2 to participate in the CHA cycle again. Ultimately, a significant amount of signal label is retained on the electrode by hybridizing with sheared HP1, generating a robust electrical signal. SignificanceThrough the signal amplification strategy, the aptasensor design provides a broad linear range of 0.005–500 nM, with a low detection limit of 0.112 pM for TOB. It is worth mentioning that the aptasensor displayed favorable stability, specificity, and reproducibility, and has been successfully applied to practical samples, demonstrating its utility in practical applications.