A novel electrochemical sensing strategy for rapid and ultrasensitive detection of Salmonella by rolling circle amplification and DNA–AuNPs probe

A novel electrochemical sensing strategy was developed for ultrasensitive and rapid detection of Salmonella by combining the rolling circle amplification with DNA–AuNPs probe. The target DNA could be specifically captured by probe 1 on the sensing interface. Then the circularization mixture was added to form a typical sandwich structure. In the presence of dNTPs and phi29 DNA polymerase, the RCA was initiated to produce micrometer-long single-strand DNA. Finally, the detection probe (DNA–AuNPs) could recognize RCA product to produce enzymatic electrochemical signal. Under optimal conditions, the calibration curve of synthetic target DNA had good linearity from 10 aM to 10 pM with a detection limit of 6.76 aM (S/N = 3). The developed method had been successfully applied to detect Salmonella as low as 6 CFU mL⁻¹ in real milk sample. This proposed strategy showed great potential for clinical diagnosis, food safety and environmental monitoring.     

G-quadruplex − based homogenous fluorescence platform for ultrasensitive DNA detection through isothermal cycling and cascade signal amplification

We describe a simple and homogenous fluorimetric method for sensitive determination of DNA. It is based on target-triggered isothermal cycling and a cascade exponential amplification reaction that generates a large amount of a G-quadruplex. This results in strong fluorescence signal when using thioflavin T as a G-quadruplex-specific light-up fluorescent probe. Tedious handling after amplification is widely eliminated by the addition of thioflavin T. No other exogenous reagent is required. This detection platform is inexpensive and rapid, and displays high sensitivity for target DNA, with a detection limit as low as 91 pM.

The addition of target DNA can trigger the isothermal exponential amplification reaction to generate a large amount of G-quadruplex sequence oligonucleotides and then employ thioflavin T (Th T) (a G-quadruplex-specific light-up dye) as signal output for sensitive DNA detection.

     

An electrochemical aptasensor based on cu2+-induced signal amplification strategy for the detection of ochratoxin aEI北大核心CSCD

A novel electrochemical aptasensor based on a Cu2+-induced signal amplification strategy was constructed for the rapid, sensitive and specific detection of ochratoxin A （OTA）. The OTA aptamer with poly （T） was hybridized with the captured DNA probe on the electrode surface. In the presence of Cu2+ and ascorbic acid, the end of poly（T） was used as a template to in situ grow copper nanoclusters （Cu NCs）. In the absence of targeted OTA, the gold electrodes after decorating Cu NCs were immersed into an acidic environment to release Cu2+. After enriching Cu2+ at a potential of − 1.6 V, the strongest current value of copper was recorded by measuring differential pulse voltammetry （DPV）. In the presence of OTA, the OTA aptamer was tightly bound to the target OTA. The OTA aptamer broke away from the electrode to reduce the growth of Cu NCs, resulting in lower DPV current response. This proposed method was employed to detect OTA with linear range from 0.1 to 50.0 ng/mL, and the detection limit was 41.2 pg/mL. The Cu2+-induced electrochemical aptasensor can be further applied in the analysis of target OTA in coffee solution samples. © 2023, Youke Publishing Co.,Ltd. All rights reserved.     

A novel aptasensor strategy for protein detection based on G-quadruplex and exonuclease Ⅲ-aided recycling amplification

The detection of biomarkers is of great significance in the diagnosis of numerous diseases,especially cancer.Herein,we developed a sensitive and universal fluorescent aptasensor strategy based on magnetic beads,DNA G-quadruplex,and exonuclease III(Exo III).In the presence of a target protein,a label-free single strand DNA(ssDNA) hybridized with the aptamer was released as a trigger DNA due to specific recognition between the aptamer and target.Subsequently,ssDNA initiates the Exo Ⅲ-aided recycling to amplify the fluorescence signal,which was caused by N-methylmesoporphyrin IX(NMM)insertion into the G-quadruplex structure.This proposed strategy combines the excellent specificity between the aptamer and target,high sensitivity of the fluorescence signal by G-quadruplex and Exo Ⅲ-aided recycling amplification.We selected(50-1200 nmol/L) MUC1,a common tumor biomarker,as the proof-of-concept target to test the specificity of our aptasenso r.Results reveal that the sensor sensitively and selectively detected the target protein with limits of detection(LODs) of 3.68 and 12.83 nmol/L in buffer solution and 10% serum system,respectively.The strategy can be easily applied to other targets by simply substituting corresponding aptamers and has great potential in the diagnosis and monitoring of several diseases.     

Analyte-triggered cyclic autocatalytic oxidation amplification combined with an upconversion nanoparticle probe for fluorometric detection of copper(II)

The authors describe an upconversion nanoparticle-based (UCNP–based) fluorometric method for ultrasensitive and selective detection of Cu²⁺. The UCNPs show a strong emission band at 550 nm under near-infrared excitation at 980 nm. The principle of the strategy is that gold nanoparticles (AuNP) can quench the fluorescence of UCNP. In contrast, the addition of L-cysteine (Cys) can induce the aggregation of AuNP, resulting in a fluorescence recovery of the UCNPs. On addition of Cu²⁺, it oxidizes Cys to cystine and is reduced to Cu⁺. The Cu⁺ thusformed can be oxidized cyclically to Cu²⁺ by dissolved O₂, which catalyzes and recycles the whole reaction. Thus, the aggregation of AuNP is inhibited and the fluorescence recovered by Cys is quenched. Under the optimal condition, the quenching efficiency shows a good linear response to the concentrations of Cu²⁺ in the 0.4–40 nM range. The limit of detection is 0.16 nM, which is 5 orders of magnitude lower than the U.S. Environmental Protection Agency limit for Cu²⁺ in drinking water (20 μM). The method has been further applied to monitor Cu²⁺ levels in real samples. The results of detection are well consistent with those obtained by atomic absorption spectroscopy.

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Graphical abstract Gold nanoparticles (AuNP) as a high efficient fluorescence quenching reagent of upconversion nanoparticles (UCNP) were used in a fluorometric method for detection of Cu²⁺ based on a cyclic catalytic oxidation amplification strategy.

     

Rolling cycle amplification based single-color quantum dots–ruthenium complex assembling dyads for homogeneous and highly selective detection of DNA

In this work, a new, label-free, homogeneous, highly sensitive, and selective fluorescent biosensor for DNA detection is developed by using rolling-circle amplification (RCA) based single-color quantum dots–ruthenium complex (QDs–Ru) assembling dyads. This strategy includes three steps: (1) the target DNA initiates RCA reaction and generates linear RCA products; (2) the complementary DNA hybridizes with the RCA products to form long double-strand DNA (dsDNA); (3) [Ru(phen)₂(dppx)]²⁺ (dppx = 7,8-dimethyldipyrido [3,2-a:2′,3′-c] phenanthroline) intercalates into the long dsDNA with strong fluorescence emission. Due to its strong binding propensity with the long dsDNA, [Ru(phen)₂(dppx)]²⁺ is removed from the surface of the QDs, resulting in restoring the fluorescence of the QDs, which has been quenched by [Ru(phen)₂(dppx)]²⁺ through a photoinduced electron transfer process and is overlaid with the fluorescence of dsDNA bonded Ru(II) polypyridyl complex (Ru-dsDNA). Thus, high fluorescence intensity is observed, and is related to the concentration of target. This sensor exhibits not only high sensitivity for hepatitis B virus (HBV) ssDNA with a low detection limit (0.5 pM), but also excellent selectivity in the complex matrix. Moreover, this strategy applies QDs–Ru assembling dyads to the detection of single-strand DNA (ssDNA) without any functionalization and separation techniques.     

An electrochemical biosensor based on DNA “nano-bridge” for amplified detection of exosomal microRNAs

Exosomal miRNAs, as potential biomarkers in liquid biopsy for cancer early diagnosis, have aroused widespread concern. Herein, an electrochemical biosensor based on DNA “nano-bridge” was designed and applied to detect exosomal microRNA-21 (miR-21) derived from breast cancer cells. In brief, the target miR-21 can specifically open the hairpin probe 1(HP1) labeled on the gold electrode (GE) surface through strand displacement reaction. Thus the exposed loop region of HP1 can act as an initiator sequence to activate the hybridization chain reaction (HCR) between two kinetically trapped hairpin probes: HP2 immobilized on the GE surface and biotin labeled HP3 in solution. Cascade HCR leads to the formation of DNA “nano-bridge” tethered to the GE surface with a great deal of “piers”. Upon addition of avidin-modified horseradish peroxidase (HRP), numerous HRP were bound to the formed “nano-bridge” through biotin-avidin interaction to arouse tremendous current signal. In theory, only a single miR-21 is able to trigger the continuous HCR between HP2 and HP3 until all of the HP2 are exhausted. Therefore the proposed biosensor achieved ultrahigh sensitivity toward miR-21 with the detection limit down to 168 amol/L, as well as little cross-hybridization even at the single-base-mismatched level. Successful attempts were also made in the detection of exosomal miR-21 obtained from the MCF-7 of breast cancer cell line. To our knowledge, this is the first attempt to built horizontal DNA nano-structure on the electrode surface for exosomal miRNAs detection. In a word, the high sensitivity, selectivity, low cost make the proposed method hold great potential application for early point-of-care (POC) diagnostics of cancer.     

A novel turn-on fluorometric “reporter-spacer-receptor” chemosensor based on calix[4]arene scaffold for detection of cyanate anion

Herein, a novel calix[4]arene compound, which was modified by the 2-(2-aminophenyl)benzothiazole fragment with cyanate recognition function was designed based on the reporter-spacer-receptor sensing system. The construction was done via two-step reaction, and the desired sensor 4 was characterized by FT-IR, ¹H-, ¹³C-NMR, and fluorescence spectroscopy along with HRMS data. The sensor candidate showed distinct fluorometric cyanate detection by means of reporter feature of selected benzothiazole constituent. In the presence of cyanate, the sensor gave a turn-on-type fluorescence at 482 nm with a large stokes' shift. Furthermore, it was observed that our fluoroionophore 4 is highly selective toward cyanate over remaining anions such as sulfate, phosphate, fluoride, chloride, bromide, iodide, chlorate, and nitrate in 10% aqueous solution of DMSO. The 1:2 stoichiometric ratio of the 4 -cyanate complex was given the best fit with Job's plot based on the titration data. The association constant (K_a) of sensor 4 with cyanate is determined to be 1.64 × 10⁵ M⁻². The obtained limit of detection (LOD) value for cyanate anion, 312 nM, clearly revealed the remarkable sensitivity of the chemosensor 4 . This supramolecular method provides a highly adaptive technique for the detection of cyanate and so cyanide ions by current international standard methods.     

An electrochemical signal switch–based (on–off) aptasensor for sensitive detection of insulin on gold-deposited screen-printed electrodes

Journal of Solid State Electrochemistry - Insulin hormone is of great importance for many diseases, especially for diabetes management. Therefore, different detection strategies have been used for...     

A study on the mechanism of reaction between BNAH and chloranil:Evidence for electron transfer

The reaction between chloranil and N-benzyldihydronicotinamide(BNAH)in boratebuffer/DMF was investigated.The reaction mixture gave a strong esr signal,which is consistentwith that of chloranil anion radical,and tetrachlorohydrophenol(QH_2)and N-benzylnicotinamide(BNA~+)were obtained as the sole products.When the reaction was run in benzene solution,a greencoloured charge-transfer complex between the reactants could be isolated,which decomposed in polarsolvents to give BNA-+ and QH_2.Based on kinetic studies by esr spectroscopy by the stopped-flowtechnique,a two-step electron-transfer mechanism for the reactionis proposed in contrast to thehydride-transfer mechanism reported in the literature.     

A resonance light scattering sensor based on methylene blue–sodium dodecyl benzene sulfonate for ultrasensitive detection of guanine base associated mutations

A resonance light scattering (RLS) sensor for guanine base associated mutations has been developed on the basis of the high selectivity of methylene blue (MB) for guanine bases in the presence of sodium dodecyl benzene sulfonate (SDBS). MB, when bound to SDBS, underwent a dramatic enhancement of its RLS intensity. However, the addition of double-stranded DNA (dsDNA) and single-stranded DNA (ssDNA) caused the strong RLS intensity of MB-SDBS to decrease, and the RLS intensity of MB-SDBS-ssDNA was much lower than that of MB-SDBS-dsDNA. Consequently, it can be concluded that the binding abilities of MB-SDBS with ssDNA and dsDNA were different. Besides, the experimental results showed that MB-SDBS could bind specifically to oligonucleotides rich in guanine bases. Short DNA targets with sequences related to β-thalassaemia, thrombophilia and psoriasis, all of which are guanine base relevant mutations, were synthesized. It was found that MB-SDBS could recognize the single-base mismatches in the mutational DNA, followed by different RLS signal changes between MB-SDBS-normal DNA systems and MB-SDBS-mutational DNA systems. The ultrasensitive sensor allows simple, rapid, sensitive and selective detection of guanine base associated mutations, indicating its potential application in the medical field.     

A multifunctional probe based on the use of labeled aptamer and magnetic nanoparticles for fluorometric determination of adenosine 5’-triphosphate

A multifunctional fluorescent probe is synthesized for the determination of adenosine 5′-triphosphate (ATP). The 6-carboxyfluorescein-labeled aptamer (FAM-aptamer) was bound to the surface of magnetite nanoparticles coated with polydopamine (Fe₃O₄@PDA) by π-π stacking interaction to form the multifunctional probe. The probe has three functions including recognition, magnetic separation, and yielding a fluorescent signal. In the presence of ATP, FAM-aptamer on the surface of the probe binds to ATP and returns to the solution. Thus, the fluorescence of the supernatant is enhanced and can be related to the concentration of ATP. Fluorescence intensities were measured at excitation/emission wavelengths of 494/526 nm. Response is linear in the 0.1–100 μM ATP concentration range, and the detection limit is 89 nM. The probe was applied to the quantitation of ATP in spiked human urine and serum samples, with recoveries ranging between 94.8 and 102%.

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Graphical abstract A multifunctional fluorescent probe based on the use of FAM-aptamer and Fe₃O₄@PDA is described for the determination of ATP in spiked human urine and serum samples. FAM-aptamer: 6-carboxyfluorescein-labeled aptamer; Fe₃O₄@PDA: magnetite nanoparticles coated with polydopamine. ATP: adenosine 5′-triphosphate.

     

Highly sensitive fluorescent sensor for mercury ion based on photoinduced charge transfer between fluorophore and π-stacked T-Hg(II)-T base pairs

A novel and simple oligodeoxyribonucleotide-based sensor with single fluorophore-labeled for mercury ion sensing was reported. An oligodeoxyribonucleotide poly(dT) was labeled with fluorescein as donor. Based on the specific binding of Hg(II) to T-T mismatch base pairs, the formation of π-stacked [T-Hg(II)-T] with “sandwich” structure on the addition of Hg(II) ions facilitates the electron transfer via photoinduced charge transfer (PCT), which creates an additional nonradiative decay channel for excited fluorophore and triggers the fluorescence to be quenched. The π-stacked [T-Hg(II)-T] functioned not only as mercury ion recognition but also as an electron acceptor to quench the donor. A linear relationship was observed over the range of 0-1.0 μM with the detection limit of 20 nM for mercury ions. The fluorescence quenching phenomenon and quenching mechanism, reliability and selectivity of the system were investigated in detail.     

Immobilization and hybridization of DNA based on magnesium ion modified 2,6-pyridinedicarboxylic acid polymer and its application for label-free PAT gene fragment detection by electrochemical impedance spectroscopy

A new approach for a simple electrochemical detection of PAT gene fragment is described. Poly(2,6-pyridinedicarboxylic acid) (PDC) modified glassy carbon electrode (GCE) was prepared by potential scan electropolymerization in an aqueous solution. Mg2 ions were incorporated by immer-sion of the modified electrode in 0.5 mol/L aqueous solution of MgCl2 to complete the preparation of a generic "activated" electrode ready for binding the probe DNA. The ssDNA was linked to the conduct-ing polymer by forming a bidentate complex between the carboxyl groups on the polymer and the phosphate groups of DNA via Mg2 . DNA immobilization and hybridization were characterized with dif-ferential pulse voltammetry (DPV) by using methylene blue (MB) as indicator and electrochemical im-pedance spectroscopy (EIS). The EIS was of higher sensitivity for DNA detection as compared with voltammetric methods in our strategy. The electron transfer resistance (Ret) of the electrode surface in EIS in [Fe(CN)6]3-/4- solution increased after the immobilization of the DNA probe on the Mg/PDC/GCE electrode. The hybridization of the DNA probe with complementary DNA (cDNA) made Ret increase further. The difference between the Ret at ssDNA/Mg/PDC/GCE and that at hybridization DNA modified electrode (dsDNA/Mg/PDC/GCE) was applied to determine the specific sequence related to the target PAT gene with the dynamic range comprised between 1.0 × 10-9 and 1.0 × 10_5 mol/L. A detection limit of 3.4 × 10-10 mol/L of oligonucleotides can be estimated.     

A novel colorimetric aptasensor for detection of chloramphenicol based on lanthanum ion–assisted gold nanoparticle aggregation and smartphone imaging

A label-free, rapid response colorimetric aptasensor for sensitive detection of chloramphenicol (CAP) was proposed, which was based on the strategy of ssDNA-modified gold nanoparticle (AuNP) aggregation assisted by lanthanum (La³⁺) ions. The AuNPs generated a color change that could be monitored in the red, green, and blue and analyzed by the smartphone imaging app. La³⁺, as a trigger agent, strongly combined with the phosphate groups of the surface of ssDNA-AuNPs probe, which helps create AuNP aggregation and the color change of AuNPs from red to blue. On the contrary, when mixing with CAP, the aptamer (Apt) bound to CAP to form a rigid structure of the Apt-CAP complex, and La³⁺ attached to the phosphate groups of the complex, which prevented the aptamer from binding to the surface of the AuNPs. As a result, the color of the AuNPs changed to violet-red. Finally, UV-vis absorption spectroscopy and the smartphone imaging app were employed to determine CAP with a lower detection limit of 7.65 nM and 5.88 nM, respectively. The proposed strategy featuring high selectivity and strong anti-interference ability for detection of CAP in practical samples was achieved. It is worth mentioning that the simple and portable colorimetric aptasensor will be used for facilitating on-site detection of food samples.

     

Portable biosensor for quantitative detection of melamine based on CRISPRCas12a and a personal glucose meterEI北大核心CSCD

A CRISPR-Cas system holds great promise as a next-generation biosensing technology for molecular diagnostics. In this paper, a portable biosensor based on the trans-cleavage activity of CRISPR-Cas12a and a personal glucose meter has been developed for quantitative, sensitive and specific detection of melamine. The presence of the target melamine binds to the aptamer, leading to the release of locker DNA. And then, the leasing locker DNA activates the trans-cleavage activity of CRISPR-Cas12a to cleave the single-strand DNA (ssDNA) linker on sucrase-ssDNA modified electrode, releasing a short DNA fragment labeled with sucrase in the resulting solution. The sucrase could further catalyze sucrose to glucose, which could be detected by the PGM. Under the optimized conditions, the increase of PGM signal was relative with the concentration of melamine ranging from 0.1 to 2.5 μmol/L and the limit of detection (LOD) was 37 nmol/L. Moreover, the portable biosensor has strong specificity and can be used for the quantitative detection of melamine in milk samples. © 2023, Youke Publishing Co.,Ltd. All rights reserved.