Dual Amplification Strategy Research Articles

Electrochemical detection of MMP-2 with enhanced sensitivity: Utilizing T7 RNA polymerase and CRISPR-Cas12a for neuronal studies

This study introduces a novel electrochemical biosensor for detecting Matrix Metalloproteinase-2 (MMP-2), a key biomarker in cancer diagnostics and tissue remodeling. The biosensor is based on a dual-amplification strategy utilizing T7 RNA polymerase isothermal amplification and CRISPR-Cas12a technology. The principle involves the release of a DNA template in the presence of MMP-2, leading to RNA synthesis by T7 RNA polymerase. This RNA activates CRISPR-Cas12a, which cleaves a DNA probe on the electrode surface, resulting in a measurable electrochemical signal.The biosensor demonstrated exceptional sensitivity, with a detection limit of 2.62 fM for MMP-2. This high sensitivity was achieved through the combination of transcriptional amplification and the collateral cleavage activity of CRISPR-Cas12a, which amplifies the signal. The sensor was able to detect MMP-2 across a wide dynamic range from 2 fM to 1 nM, showing a strong linear correlation between MMP-2 concentration and the electrochemical signal. In practical applications, the biosensor accurately detected elevated levels of MMP-2 in cell culture supernatants from HepG2 liver cancer cells, distinguishing them from normal LO2 liver cells. The use of an MMP-2 inhibitor confirmed the specificity of the detection. These results underscore the biosensor's potential for clinical diagnostics, particularly in early cancer detection and monitoring of tissue remodeling activities. The biosensor's design allows for rapid, point-of-care testing without the need for complex laboratory equipment, making it a promising tool for personalized healthcare and diagnostic applications.

Highly sensitive ECL detection of miRNA based on self-generated coreactant and target-induced catalyzed hairpin assembly

BackgroundRecently, various techniques have been developed to accurately and sensitively detect tumor biomarkers for the early diagnosis and effective therapy of cancer. The electrochemiluminescence (ECL) method holding outstanding features including high sensitivity, ease of operation, and spatiotemporal controllability exhibited great potential for DNA/RNA detection, immunoassay, cancer cell detection, and environmental analysis. However, a glaring problem of ECL approaches is that the layer-by-layer modification on the electrode leads to poor stability and sensitivity of the sensors. Therefore, new simple and efficient methods for electrode modification which can effectively improve the ECL signal have attracted more and more research interests. ResultsBased on the dual amplification strategy of target-induced CHA and nanocomposite probes leading to self-generated co-reactant (H2O2), we proposed a highly sensitive miRNA-ECL detection system. The introduction of the target miRNA-21 triggers the CHA cycle amplification of DNA1 and biotin-modified DNA2, releasing the target miRNA-21 sequence for the target cycle reaction. After the reaction, the newly introduced DNA2 was combined with Au NPs modified with SA and Glucose oxidase (GOD). In the presence of oxygen, glucose was decomposed by GOD to produce H2O2, and then H2O2 was immediately catalyzed by the Hemin/G-quadruplex at the double-stranded end of the CHA product to produce a large amount of O2−•. As a co-reactant of luminol, the ECL signal was significantly enhanced, thereby achieving highly sensitive detection of miRNA-21 content and obtaining a low detection limit of 0.65 fM. The high specificity of the ECL biosensor was also proved by base mismatch. SignificanceCompared with other current detection methods, this sensor can achieve quantitative analysis of other target analytes by flexibly changing the probe DNA sequence, and provide a new feasible solution for the detection of tumor-associated markers. Benefiting from the improved sensitivity and selectivity, the proposed biosensing platform is expected to provide a new strategy for biomarkers analysis and outstanding prospect for further clinical application.

An electrochemical biosensor with homogeneous dual amplification strategy for sensitive analysis of Pb2+

Herein, a homogeneous double amplified electrochemical biosensing system was designed on the basis of rolling circle amplification (RCA) and DNAzyme-assisted Pb2+ recycling amplification with the assistance of the signal molecule methylene blue (MB), which was used for highly sensitive analysis of Pb2+. In comparison with the conventional electrochemical biosensor, the homogeneous strategy adopted in this work can avoid the complex electrode modification and immobilization processes, and address the issues of the large steric hindrance and the restricted probe freedom on the sensing interface, thus achieving high recognition efficiency between recognition unit and target molecule as well as excellent detection performance of biosensor. In addition, the double amplified method assembling RCA and DNAzyme-assisted Pb2+ recycling amplification can convert a handful of target Pb2+ into abundant signal molecule MB, which would lead to the double amplification of output signal and the significantly enhanced detection sensitivity. Taking advantages of homogeneous dual amplification strategy, the constructed electrochemical biosensor for Pb2+ analysis emerged a good linear range of 50 fM to 500 nM and a detection limit of 16.7 fM with significant selectivity and desirable stability. Impressively, the result presented herein comprise a valuable reference for establishing homogeneous multiple amplification model and constructing innovative biosensing system for sensitive detection of heavy metal ions.

Highly sensitive and label-free immunoassay of chloramphenicol based on poly-adenine-mediated spherical nucleic acid and hybridization chain reaction amplification

In this paper, a label-free fluorescent immunoassay for chloramphenicol was established based on a DNA-assisted signal amplification strategy. The poly-adenine mediated spherical nucleic acid was firstly employed as the substrate of antibody to construct the immunonanoprobes. Enzyme-free signal amplification techniques, hybridization chain reaction and strand displacement reaction, were utilized to develop the dual amplification strategy. The output signal was expressed through the use of G-quadruplex to achieve label free fluorescence changes. Under optimized conditions, the detection limit of this method was reduced to 0.0044 ng/L. The method exhibited a high accuracy to the detection of chloramphenicol in milk samples with a recovery rate from 94.5% to 118.1%. However, the limitations of this method probably lied in the overlong detection time and the possible interference from other compounds present in food samples. The developed label-free fluorescent immunoassay could provide an innovative view for the reliable and ultrasensitive detection of chloramphenicol, which is conducive to the food safety.

Low-triggering-potential electrochemiluminescence based on mental-organic frameworks encapsulation of ruthenium for synthetic cathinone detection by coupling photonic crystal light-scattering signal amplification of covalent-organic frameworks

Developing effective electrochemiluminescence (ECL) platforms is always an essential concern in highly sensitive bioanalysis. In this work, a low-triggering-potential ECL sensor was designed for detecting synthetic cathinone 3,4-methylenedioxypyrovalerone (MDPV) based on a dual-signal amplification strategy. Initially, a probe was created by integrating Ruthenium into the hollow porphyrin-based MOF (PCN-222) structure to decrease the excitation potential and enhance ECL performance without external co-reaction accelerators. Additionally, for the first time, photonic crystals (PCs) assembled from covalent organic frameworks (COFs) were employed to amplify the ECL signal, thereby increasing the photon flux and the loading capacity of the ECL emitter to enhance sensitivity of the sensor. In the presence of the target MDPV, the aptamer labeled with Ferrocene (Fc) experienced conformational changes, causing Fc to approach the luminophore and resulting in ECL quenching. This effect was attributed to aptamer's conformational changes induced by the target, directly correlating with the target concentration. The constructed sensor showed good linearity with the target MDPV concentration, covering a dynamic range from 1.0 × 10−14 to 1.0 × 10−6 g/L and achieved an ultra-low detection limit of 4.79 × 10−15 g/L. This work employed dual amplification strategies to enhance ECL signals effectively, providing a novel method for developing highly responsive and bioactive sensors.

Fluorescent aptasensor for highly sensitive detection of Staphylococcus aureus based on dual-amplification strategy by integrating DNA walking and hybridization chain reaction

Food-borne diseases caused by bacteria threaten human health. Herein, we presented a new fluorescent aptasensor by coupling DNA walking and hybridization chain reaction (HCR) for convenient and sensitive quantification of bacteria. Staphylococcus aureus (S. aureus) was selected as target. When there was target in the system, the binding of S. aureus with its aptamer caused the disintegration of aptamer/DNA walker on the surface of AuNPs and released DNA walker. With the help of Nt.BsmAI, DNA walker moved along the surface of AuNPs and trigger probe was detached from AuNPs. The trigger probe could initiate hybridization chain reaction (HCR) and opened the stems of H1@AuNPs probe and H2@AuNPs probe. After the addition of nicking endonuclease, the adjacent upconversion nanoparticles (UCNPs, NaYF4:Yb3+, Er3+) were further away from the quenchers (AuNPs) of H1 and H2. Therefore, the fluorescence intensity of UCNPs could be restored via fluorescence resonance energy transfer (FRET). Bacteria were thus detected by recording the fluorescence intensity of UCNPs. This method is simple, rapid and sensitive. It can directly detect bacteria in a low background signal. The limit of detection (LOD) was 10 CFU/mL, detection time was less than 3 h. Recovery rates in simulated milk, honey and human serum samples ranged from 93.6 % to 105.8 %. The strategy opens up new paths for early diagnosis of diseases and food monitoring.

An electrochemical DNA biosensor coupled with dual amplification strategy based on DNA-Au bio-bar codes and silver enhancement for highly sensitive and selective detection of MDR1 gene

Given the tumors might develop multidrug resistance (MDR) during chemotherapy that leading to treatment failure, rapid and sensitive determination of MDR1 gene is an effective tool to monitor the development of MDR. Herein, we established an electrochemical deoxyribonucleic acid (DNA) biosensor coupled with dual amplification strategy based on DNA-Au bio-bar codes and enhancement of silver for sensitive and selective determination of MDR1 gene. Specifically, DNA-Au bio-bar codes as signal probe were prepared by immobilizing complementary and non-complementary sequences on gold nanoparticles (AuNPs), so as to reduce the cross reaction between the target and reporter DNA. In addition, AuNPs-catalyzed reduction of silver ions greatly amplified the current signal. Under the optimal conditions, the current signal was proportional to the base-10 logarithm of target DNA concentration in a wide range from 100 fM to 1 nM with a detection limit as low as 33 fM (S/N = 3). Moreover, without the need for target amplification, the prepared biosensor exhibited superior selectivity and high sensitivity even in the presence of other sequences. Therefore, the developed electrochemical DNA biosensor is a facile and sensitive platform for MDR1 gene detection.

Green synthesized gold nanoparticles and CuO-based nonenzymatic sensor for saliva glucose monitoring.

Glucose, essential for brain and muscle functions, requires careful monitoring in diabetes and other chronic disease management. While blood glucose monitoring provides precise information about these diseases, it remains an invasive method. Saliva glucose monitoring could offer an alternative approach, but the glucose concentration in saliva is very low. In this work, we report a simple, low-cost, highly sensitive nonenzymatic electrochemical glucose sensor. We developed this sensor using green synthesized gold nanoparticles (AuNPs) and wet chemical synthesized copper oxide (CuO) nanoparticles on a screen-printed carbon electrode (Au/CuO/SPCE). The sensor's high sensitivity results from dual amplification strategies using AuNPs and CuO nanomaterials, each demonstrating catalytic activity towards glucose. This shows promising potential for saliva glucose monitoring. The AuNPs were synthesized using an Au precursor and orange peel extract (OPE), yielding stable colloidal AuNPs with a mean diameter of about 37 nm, thus eliminating the need for additional capping agents. Under optimal conditions, amperometric tests revealed that the sensor responded linearly to glucose concentrations ranging from 2 μM to 397 μM with a sensitivity of 236.70 μA mM-1 cm-2. Furthermore, the sensor demonstrated excellent reproducibility, stability and high selectivity for glucose in the presence of different biomolecules. We validated the sensor's efficacy by measuring glucose in human saliva, showing its potential for noninvasive glucose monitoring. This research advances the development of point-of-care devices, positioning the sensor as a promising tool for noninvasive glucose monitoring and improved diabetes management.

Signal Modulation of Organic Photoelectrochemical Transistor by a Z-Scheme Photocathodic Gate: An Innovative Dual Amplification Strategy for Sensitive Aptasensing Application.

Pursuing a more efficient signal amplification strategy is highly demanded for improving the performance of the promising cathodic photoelectrochemical (PEC) sensors. In this work, we present an extremely effective dual signal amplification strategy by the integration of a Z-scheme nanohybrids-based photocathode with the effective signal modulation of an organic photoelectrochemical transistor (OPECT) device. Specifically, photocathodic gate material of CdTe-BiOBr nanohybrids with a Z-scheme electron-transfer route was designed and synthesized for preliminary improvement of the activity of the photogate; afterward, signal modulation of the OPECT system by the photocathodic gate of CdTe-BiOBr was then accomplished for further signal amplification by 2 orders of magnitude. As a result, the output PEC signal of CdTe-BiOBr was enhanced by 17.5-fold as compared to BiOBr, and the channel current (IDS) of the OPECT device was 117-fold magnified than its gate current (IG) response. Exemplified by tetracycline (TC) as a model target and aptamer as the specific recognition element, a versatile cathodic aptasensing platform was constructed based on the proposed OPECT device. The introduced OPECT aptasensor merits advantages, including a good linear range (1.0 × 10-12 to 1.0 × 10-6 M), a low limit of detection (4.2 × 10-13 M), and superior sensitivity than the traditional PEC methods for TC detection, which represents a universal protocol for developing the innovative photocathodic OPECT sensing platform toward accurate analysis.

Programmable SARS-CoV-2 mutation detection platform based on ligation-triggered isothermal exponential amplification coupled with self-priming amplification

In this work, we developed a programmable mutation detection platform with a dual amplification strategy for universal detection of SARS-CoV-2 mutations based on ligation-triggered isothermal exponential amplification reaction(L-EXPAR) and phosphorothioate modified self-priming amplification (PS-SPA). In the presence of mutant target (MT) sequence, it complements with hairpin probe (H1) and hairpin probe (H2) and triggered L-EXPAR at lower temperature. A large number of single-strand DNA (ssDNA) is produced in the circle of ligation, nicking, polymerization, and strand displacement. The ssDNA products containing target sequences can feed back and initiate another L-EXPAR. The ligated H1-H2 complex triggers another isothermal process termed PS-PSA at higher temperature. 20 bases of H1-H2 complex on the 5′ end modified with PS can form a short stem-loop hairpin due to the lower Tm of PS modification. The resulting overhanging 3′ end can also fold back as a new primer to initiate the next round of polymerization. In the circle of PS-SPA, the H1-H2 chain can be extended to be a long hairpin concatemer containing a great number of tandem target sequences. The resulting DNA products can be monitored in real-time fluorescent signaling. In the presence of the SARS-CoV-2 wild type (WT) sequence, the ligation between H1 and H2 cannot happen due to the mismatch of the last base on the 3′ end of H1. The proposed assay displayed a good linear range from 1 fM to 1 nM with a detection limit of femtomole level for the detection of SARS-CoV-2 spike gene D614G mutation and Del246-252 + D253N deletion.

A dual amplification strategy integrating entropy-driven circuit with Cas14a for sensitive detection of miRNA-10b

Cas14a may recognize and randomly cleave ssDNAs without protospacer adjacent motif (PAM) sequence restriction to benefit nucleic acid sensing. However, the sensitivity of individual Cas proteins is limited. Herein a protocol based on entropy-driven circuit (EDC) and Cas14a/sgRNA (EDC-Cas14a) was developed to achieve dual signal amplification of miRNA. The target can continuously catalyze the reaction of EDC to generate multiple ssDNA activators, which triggers the trans-cleavage of Cas14a to cleave reporters and achieves 2nd signal amplification. The background signal is effectively reduced by adjusting the number of bases in the EDC. Therefore, two parts of the protocol can coexist in a system without interfering with each other. EDC significantly improves the sensitivity and specificity of Cas14a/sgRNA. A 100-fold improvement on sensitivity was achieved with a limit of detection of 2.1 pM. The specificity of EDC-Cas14a was characterized by outstanding distinguishing capability of miRNA-10b and its counterpart with one-base mismatch. EDC-Cas14a can be readily adjusted to detect various miRNAs by simply manipulating EDC modules. Eventually, the promising assay performances of EDC-Cas14a was demonstrated by the detection of miRNA-10b in serum and cell lysate to provide a general approach for sensitive assay of miRNA biomarker in molecular diagnosis.

Profiling extracellular vesicle surface proteins with 10 µL peripheral plasma within 4h.

Extracellular vesicle (EV) surface proteins, expressed by primary tumours, are important biomarkers for early cancer diagnosis. However, the detection of these EV proteins is complicated by their low abundance and interference from non-EV components in clinical samples. Herein, we present a MEmbrane-Specific Separation and two-step Cascade AmpLificatioN (MESS2CAN) strategy for direct detection of EV surface proteins within 4h. MESS2CAN utilises novel lipid probes (long chains linked by PEG2K with biotin at one end, and DSPE at the other end) and streptavidin-coated magnetic beads, permitting a 49.6% EV recovery rate within 1h. A dual amplification strategy with a primer exchange reaction (PER) cascaded by the Cas12a system then allows sensitive detection of the target protein at 10 EV particles per microliter. Using 4 cell lines and 90 clinical test samples, we demonstrate MESS2CAN for analysing HER2, EpCAM and EGFR expression on EVs derived from cells and patient plasma. MESS2CAN reports the desired specificity and sensitivity of EGFR (AUC=0.98) and of HER2 (AUC=1) for discriminating between HER2-positive breast cancer, triple-negative breast cancer and healthy donors. MESS2CAN is a pioneering method for highly sensitive in vitro EV diagnostics, applicable to clinical samples with trace amounts of EVs.

Immobilization-free electrochemical homogeneous aptasensor for highly sensitive detection of carcinoembryonic antigen by dual amplification strategy

Electrochemical aptasensor has been widely studied, while its practical application is limited by the unavoidable variations of aptamer loading densities and low signal amplification efficiency. To overcome these restrictions, an immobilization-free and label-free electrochemical homogeneous aptasensor was constructed for carcinoembryonic antigen (CEA) assay by combining RecJf exonuclease-mediated target cycling strategy and rolling circle amplification technology. In this system, the pre-immobilization of aptamers or other relevant signal elements on the electrode substrate is no longer necessary, thus the electrochemical homogeneous aptasensor shows good versatility on different transducers. Moreover, the whole recognition and signal amplification process are activated instantaneously by a non-professional operation of the solution mixture. This strategy can not only increase the stability (95.1% after 30 days of storage) and reproducibility (2.12% among five independent electrodes), but also further improve the sensitivity (detection limit of fg mL−1 level) due to the free target recognition and dual signal amplification in the homogeneous solution phase. The proposed immobilization-free electrochemical homogeneous aptasensors on different electrode substrates both achieve satisfactory results in actual sample tests, which has the potential for commercial applications and the establishment of other target platforms in the future.

CRISPR/Cas12a-Mediated Aptasensor Based on Tris-(8-hydroxyquinoline)aluminum Microcrystals with Crystallization-Induced Enhanced Electrochemiluminescence for Acetamiprid Analysis.

Improving the electrochemiluminescence (ECL) efficiency of luminophores has always been the goal of the ECL field. Herein, a novel crystallization-induced enhanced ECL (CIE ECL) strategy was exploited to significantly enhance the ECL efficiency of metal complex tris-(8-hydroxyquinoline)aluminum (Alq3). Alq3 monomers self-assembled and directionally grew to form Alq3 microcrystals (Alq3 MCs) in the presence of sodium dodecyl sulfate. The highly ordered crystal structure of Alq3 MCs not only constrained the intramolecular rotation of Alq3 monomers to decrease nonradiative transition but also accelerated the electron transfer between Alq3 MCs and coreactant tripropylamine to increase radiative transition, thus leading to a CIE ECL effect. Alq3 MCs exhibited brilliant anode ECL emission, which was 210-fold stronger than that of Alq3 monomers. The exceptional CIE ECL performance of Alq3 MCs coupled the efficient trans-cleavage activity of CRISPR/Cas12a assisted by rolling circle amplification and catalytic hairpin assembly to fabricate a CRISPR/Cas12a-mediated aptasensor for acetamiprid (ACE) detection. The limit of detection was as low as 0.79 fM. This work not only innovatively exploited a CIE ECL strategy to enhance the ECL efficiency of metal complexes but also integrated CRISPR/Cas12a with a dual amplification strategy for the ultrasensitive monitoring of pesticides such as ACE.

A cellular NO sensor based on aggregation-induced electrochemiluminescence and photoelectron transfer of a novel ruthenium(II) complex

Here, we present a dual amplification strategy of electrochemiluminescence (ECL) based on aggregation-induced and photoelectron transfer (PET) inhibition using a designed ruthenium-based complex for the highly sensitive detection of nitric oxide (NO). A novel ruthenium(II) complex ([Ru(phen)2(DA-phen)]2+, DA-phen: 5,6-diamino-1,10-phenanthroline) was synthesized that uses the O-diaminophenyl group for specific recognition of NO and the ruthenium metal center with 1,10-phenanthroline as the luminophore. In the first step, the 1,10-phenanthroline groups function as the aggregation part. Increasing the volume fraction of a poor solution (H2O: fw, v%) in the DMSO mixture system from 0% to 80% yielded an increase in the fluorescence (FL) and electrochemiluminescence (ECL) intensities by 3- and 42-fold when compared with the FL and ECL intensities in pure DMSO, respectively. The second step involves a PET process. Reacting the complex with NO in physiological pH aqueous media under aerobic conditions to afford its triazole derivative, [Ru(phen)2(TA-phen)]2+ (TA-phen: 5,6-triazole-1,10-phenanthroline), inhibited the electron transfer process, yielding a remarkable further increase in the FL and ECL efficiencies of the Ru(II) complex. The ECL intensities was 86-fold higher in the presence of NO. This dual amplification strategy enabled the sensitive detection of NO from 1.8 × 10–10 to 1.8 × 10–5 M with a detection limit of 60 pM (S/N = 3) and offered excellent specificity with no detectable interference by RNS and metal ions. Furthermore, selective NO detection in cells was achieved using this complex. Thus, this assay should be suitable for applications in environmental toxicology research and early clinical diagnosis.

Ultrasensitive fluorescence detection of multiple DNA methyltransferases based on DNA walkers and hyperbranched rolling circle amplification

The accurate and ultrasensitive detection of multiple methyltransferases was in great request for clinical diagnosis and epigenetic therapy. Here, a novel fluorescence assay was proposed for ultrasensitive CpG methyltransferase (M.SssI) and DNA adenine methyltransferase (Dam) activity detection based on hyperbranched rolling circle amplification (HRCA) and DNA walkers. The biosensor showed an extremely high sensitivity due to the dual-amplification strategy of HRCA and DNA walker. The LOD of the biosensor for M.SssI and Dam methyltransferase was estimated at 0.0004 U/mL and 0.001 U/mL, respectively. Without the presence of M.SssI methyltransferase, the corresponding recognition site of hairpin HM was cleaved by HpaII endonuclease, generating a DNA fragment (T-DNA) and inducing the DNA walker-HRCA reaction. Since the HRCA products contained numerous double-strand DNA (dsDNA), SYBR Green I could be embedded in the dsDNA, leading to a high fluorescent signal. In the presence of M.SssI methyltransferase, the corresponding recognition site of hairpin HM was methylated and the HpaII endonuclease-catalyzed stem of hairpin HM dissociation was hindered, leading to no DNA fragment (T-DNA) present. Hence, the DNA walker-HRCA reaction was not initiated and the fluorescent signal of SYBR Green I remained at a low level. Similarly, DNA adenine methyltransferase (Dam) and its inhibitors could also be detected by redesigning hairpin HD with the Dam recognition sequences. Furthermore, the sensing system was applied to analyze the endogenic Dam methyltransferase in the real samples such as E. coli cell lysate.

MOF-based nanocomposites coupled with RecJf exonuclease-assisted target recycling amplification: Dual signal amplification for ultrasensitive detection of vanillin

A “signal off” electrochemical aptasensor based on metal-organic framework (MOF) - based nanocomposites and RecJf exonuclease-assisted target recycling was constructed for ultrasensitive detection of vanillin (VAN). In this work, benefited from the high catalytic activity of the synthesized gold nanoparticle/iron-based MOF/polyetherimide-reduced graphene oxide (AuNPs/NH2-MOF-235(Fe)/PEI-rGO) and the AuNPs/Ce-MOF nanocomposites toward thionine (Thi), the electrochemical signal was amplified. And the target recycling assisted by RecJf exonuclease were triggered with the presence of VAN, which induced the strongly reduced electrochemical signal. On account of dual amplification strategies, the performance of the aptasensor was significantly improved to achieve ultrasensitive detection of VAN. Under optimal conditions, the linear relationship was fitted for VAN with a linear range of 2 pM - 0.2 mM and the detection limit of 1.14 pM (S/N = 3). Furthermore, this assay for VAN has excellent specificity, long-term stability, and satisfactory results of the detection in the real samples. In summary, the aptasensor could be further used for the specific quantitative determination of other targets in foods.

Electrochemiluminescence detection of miRNA-21 based on dual signal amplification strategies: Duplex-specific nuclease -mediated target recycle and nicking endonuclease-driven 3D DNA nanomachine

DNA nanomachines have shown potential application in the construction of various biosensors. Here, an electrochemiluminescence biosensor for the sensitive detection of miRNA-21 were reported based on three-dimensional (3D) DNA nanomachine and duplex-specific nuclease (DSN)-mediated target recycle amplification strategy. First, the bipedal DNA walkers were obtained by DSN-mediated digestion reaction initiated by target miRNA-21.3D DNA tracks were prepared by modifying Fe3O4 magnetic beads (MBs) with ferrocene-labeled DNA (Fc-DNA). The produced DNA walkers autonomously moved along 3D DNA tracks powered by nicking endonuclease. During the movement, ferrocene-labeled DNA was cleaved, resulting in large amounts of Fc-labeled DNA fragments away from the MBs surface. Finally, the liberated Fc-labeled DNA fragments were dropped on the C-g-C3N4 modified electrode surface, leading to the quenching of C-g-C3N4 electrochemiluminescence (ECL). Benefiting from the dual amplification strategy of 3D DNA nanomachine and DSN-mediated target recycling, the developed ECL biosensor exhibited an excellent performance for miRNA-21 detection with a wide linear range of 10 fM to 10 nM and a low detection limit of 1.0 fM. This work offers a new thought for the application of DNA walkers in the construction of various biosensors.

Dual amplification strategy turns TRPM2 channels into supersensitive central heat detectors

The Ca2+ and ADP ribose (ADPR)-activated cation channel TRPM2 is the closest homolog of the cold sensor TRPM8 but serves as a deep-brain warmth sensor. To unravel the molecular mechanism of heat sensing by the TRPM2 protein, we study here temperature dependence of TRPM2 currents in cell-free membrane patches across ranges of agonist concentrations. We find that channel gating remains strictly agonist-dependent even at 40°C: heating alone or in combination with just Ca2+, just ADPR, Ca2+ + cyclic ADPR, or H2O2 pretreatment only marginally activates TRPM2. For fully liganded TRPM2, pore opening is intrinsically endothermic, due to ~10-fold larger activation enthalpy for opening (~200 kJ/mol) than for closure (~20 kJ/mol). However, the temperature threshold is too high (>40°C) for unliganded but too low (<15°C) for fully liganded channels. Thus, warmth sensitivity around 37°C is restricted to narrow ranges of agonist concentrations. For ADPR, that range matches, but for Ca2+, it exceeds bulk cytosolic values. The supraphysiological [Ca2+] needed for TRPM2 warmth sensitivity is provided by Ca2+ entering through the channel's pore. That positive feedback provides further strong amplification to the TRPM2 temperature response (Q10 ~ 1,000), enabling the TRPM2 protein to autonomously respond to tiny temperature fluctuations around 37°C. These functional data together with published structures suggest a molecular mechanism for opposite temperature dependences of two closely related channel proteins.

Portable ATP bioluminescence sensor with high specificity for live Escherichia coli O157:H7 strain synergistically enhanced by orientated phage-modified stir bar extraction and bio-proliferation

Live foodborne pathogens proliferate rapidly and do great harm to human health, which requires appropriate methods to supervise. In this work, a portable adenosine triphosphate (ATP) bioluminescence sensor with high specificity for live E. coli O157:H7 strain synergistically enhanced by orientated phage-modified stir bar extraction and bio-proliferation was developed. In brief, the selected phages were directionally immobilized on the poly(diallyldimethylammonium chloride)-modified gold stir bar as the bioreceptor. Following the simple stir bar absorptive extraction and bio-proliferation in the Luria-Bertani medium, the number of captured E. coli O157:H7 exploded. Finally, it was quantified by a portable ATP bioluminescence sensor. Benefitting from the high specificity of phage and simple signal dual-amplification strategy, the proposed biosensor achieved the recognition of live bacteria at strain level with superior sensitivity. Also, the portable signal readout made it suitable for on-site detection. Under optimal conditions, this bioassay provided a detectable range of 102–107 CFU mL−1 with a low detection limit of 30 CFU mL−1 within 30 min. The detection results for real samples demonstrated that there were no differences between the assay and the plate counting method, while the detection time was largely shortened. Furthermore, the assay gives a novel path for the point-of-care test (POCT) of live E. coli strain, which is promising to be extended to other virulent strains measurement with corresponding phages.