fM Detection Limit Research Articles

High-Sensitivity Homogeneous Detection of miRNA-155 Governed by DNA Walker-Regulated Surface DNA Density of Magnetic Electrochemiluminescence Nanoparticles.

Homogeneous electrochemiluminescence (ECL) has gained attention for its simplicity and stability. However, false positives due to solution background interference pose a challenge. To address this, magnetic ECL nanoparticles (Fe3O4@Ru@SiO2 NPs) were synthesized, offering easy modification, magnetic separation, and stable luminescence. These were utilized in an ECL sensor for miRNA-155 (miR-155) detection, with locked DNAzyme and substrate chain (mDNA) modified on their surface. The poor conductivity of long-chain DNA significantly impacts the conductivity and electron transfer capability of Fe3O4@Ru@SiO2 NPs, resulting in weaker ECL signals. Upon target presence, unlocked DNAzyme catalyzes mDNA cleavage, leading to shortened DNA chains and reduced density. In contrast, the presence of short-chain DNA has minimal impact on the conductivity and electron transfer capability of Fe3O4@Ru@SiO2 NPs. Simultaneously, the material surface's electronegativity decreases, weakening the electrostatic repulsion with the negatively charged electrode, resulting in the system detecting stronger ECL signals. This sensor enables homogeneous ECL detection while mitigating solution background interference through magnetic separation. Within a range of 100 fM to 10 nM, the sensor exhibits a linear relationship between ECL intensity and target concentration, with a 26.91 fM detection limit. It demonstrates high accuracy in clinical sample detection, holding significant potential for clinical diagnostics. Future integration with innovative detection strategies may further enhance sensitivity and specificity in biosensing applications.

CRISPR/Cas12a Collateral Cleavage-Driven Transcription Amplification for Direct Nucleic Acid Detection.

The clustered regularly interspaced short palindromic repeat/Cas (CRISPR/Cas) system is a powerful tool for nucleic acid detection owing to specific recognition as well as cis- and trans-cleavage capabilities. However, the sensitivity of CRISPR/Cas-based diagnostic approaches is determined by nucleic acid preamplification, which has several limitations. Here, we present a method for direct nucleic acid detection without preamplification, by combining the CRISPR/Cas12a system with signal enhancement based on light-up RNA aptamer transcription. We first designed two DNA templates to transcribe the light-up RNA aptamer and kleptamer (Kb) RNA: the first DNA template encodes a Broccoli RNA aptamer for fluorescence signal generation, and the Kb DNA template comprises a dsDNA T7 promoter sequence and an ssDNA sequence that encodes an antisense strand for the Broccoli RNA aptamer. Hepatitis B virus (HBV) target recognition activates a CRISPR/Cas12a complex, leading to the catalytic cleavage of the ssDNA sequence. Transcription of the added Broccoli DNA template can then produce several Broccoli RNA aptamer transcripts for fluorescence enhancement. The proposed strategy exhibited excellent sensitivity and specificity with 22.4 fM detection limit, good accuracy, and stability for determining the target HBV dsDNA in human serum samples. Overall, this newly designed signal enhancement strategy can be employed as a universal sensing platform for ultrasensitive nucleic acid detection.

Target-induced activation of dual DNAzyme recycling amplifications for sensitive electrochemical fat mass and obesity-associated protein assay

The fat mass and obesity-associated protein (FTO) is vital for modulating dynamic reversible process of N6-methyladenosine (m6A) level in the nucleus and the abnormal expression of FTO correlates with generation and development of Alzheimer's disease, obesity and several cancers. The sensitive detection of FTO is therefore of significance for diagnosis of FTO-related diseases. In this work, on the basis of the demethylation enzyme capability of FTO and the dual Mg2+-dependent DNAzyme recycled signal magnification strategy, establishment of a highly sensitive electrochemical FTO biosensor is described. The target FTO molecules catalyze the demethylation of m6A-modified sequences to form effective cleavage substrates for the MboI endonuclease, leading to the release of active DNAzyme sequences to trigger subsequent dual DNAzyme recycling cycles. Many methylene blue-tagged ssDNA sequences can thus be yielded via the DNAzyme amplifications and captured on the sensor electrode to generate highly amplified currents for detecting FTO in range of 0.04 pM to 10 nM with 6.11 fM detection limit. Additionally, the sensor demonstrates high selectivity and is able to be used for detecting cell lysate FTO, showing its potential for the diagnosis and study of FTO-related diseases for various application purposes.

Multi-signal amplification electrochemiluminescence biosensor based on GO-CDTPA-AuNPs composites and PET-RAFT for ultrasensitive detection of KRAS G12C mutation

The detection of the Kirsten Rat Sarcoma (KRAS) G12C point mutation plays an important role in the early diagnosis and individualized treatment of non-small cell lung cancer (NSCLC). Herein, a novel electrochemiluminescence (ECL) biosensor for the detection of KRAS G12C mutation via GO-CDTPA-AuNPs composites and photo-induced electron/energy transfer reversible addition-fragment chain transfer (PET-RAFT) multi-signal amplification strategy was fabricated for the first time. In this work, gold nanoparticles (AuNPs) were loaded onto the graphene oxide (GO) surface to form a luminol electrochemiluminescence enhanced composite by simple amide reaction and electrostatic adsorption. Specifically, in the presence of KRAS G12C mutation DNA, i.e. target DNA (tDNA), self-assembled hairpin DNA (hDNA) on the biosensor electrode surface was activated after hybridization with the tDNA to expose a 3′-terminal amino group, that could be further linked to GO-CDTPA-AuNPs composites. To increase luminol-based ECL, GO-CDTPA-AuNPs composites contained a lot of photo-induced electron/energy transfer reversible addition-fragment chain transfer (PET-RAFT) agent loaded onto GO surface. PET-RAFT polymerization of N-acryloxysuccinimide (NAS) monomer was initiated to form polymer chains containing multiple binding sites for luminol. Due to the amplification of luminol attachment sites, very high quantities of luminol could be introduced onto the biosensor electrode surface, resulting in significantly amplified ECL signal intensity. Under optimal biosensor fabrication conditions, a good linear relationship between ECL signal intensity and the logarithm of tDNA concentration was observed over the range of 1 fM to 10 nM with a 0.12 fM limit of detection. Moreover, this strategy exhibited high selectivity and excellent applicability for KRAS G12C mutation detection in the clinical human serum samples. Thus, the fabricated biosensor has great potential for application in the clinical diagnosis.

Abstract 3794: Sensitive detection of microRNA biomarkers of pancreatic cancer using one-pot endonucleolytically exponentiated rolling circle amplification by CRISPR-Cas12a

Abstract While miRNAs offer a promising source for developing diagnostic cancer biomarkers, the progress towards clinical utilities remains largely limited, due in part to the long-standing challenge in sensitive, specific, and robust detection of miRNAs in human biofluids. Emerging next-generation molecular technologies, such as the CRISPR-based methods, promise to transform nucleic acid testing. The prevailing strategy used in existing CRISPR-based methods is to orthogonally hyphenate two separate reactions for pre-amplification, e.g., rolling circle amplification (RCA), and amplicon detection by Cas12a/13a trans-cleavage in tandem. Thus, existing CRISPR-based miRNA assays require multiple manual steps and lack the analytical performance of the gold standard, RT-qPCR. Radically deviating from the existing strategies, we developed a one-step, one-pot isothermal miRNA assay termed “Endonucleolytically eXponenTiated Rolling circle Amplification with the dual-functional CRISPR-Cas12a” (EXTRA-CRISPR) for miRNA detection with RT-qPCR-like analytical performance, including high sensitivity with a single-digit fM detection limit, single-nucleotide specificity, and rapid and flexible turnaround (from 20 min to 3 h for the entire analysis depending on targets and samples). We adapted the EXTRA-CRISPR assay to quantifying miRNA biomarkers in extracellular vesicles (EVs), a major carrier of miRNAs in human biofluids, for liquid biopsy diagnosis of pancreatic ductal adenocarcinoma (PDAC). Using the EXTRA-CRISPR, we demonstrated highly sensitive and specific profiling of a panel of four miRNA markers (miR-21, miR-196a, miR-451a, and miR-1246) in plasma EVs (PDAC patients (n = 20) and healthy donors (n = 15)). Based on the individual EV-miRNA tests (AUC values ranging from 0.677 for miR-196a to 0.793 for miR-451a), an EV signature (EV-Sig) combining miR-21, miR-451a and miR-1246 was devised with a machine learning method to improve the diagnostic performance for PDAC (AUC = 0.853). The diagnostic performance of the EXTRA-CRSPR tests were rigorously validated by parallel RT-qPCR analysis of the same clinical samples, and the RT-qPCR signature combining the three markers confers a similar AUC of 0.874 for PDAC detection. Overall, the analytical and diagnostic performance of our method were shown to be comparable with that of the commercial RT-qPCR assays, while greatly simplifying the analysis workflow. Therefore, we envision that our technology provides a promising tool to advance miRNA analysis and clinical marker development for liquid biopsy-based cancer diagnosis and prognosis. Moreover, this simple and robust assay permitted direct coupling with a 3D printed smartphone-based portable device and the lateral flow assay for signal readout, demonstrating its potential adaptability to low-cost point-of-care diagnostics. Citation Format: He Yan, Song Han, Steven Hughes, Yong Zeng. Sensitive detection of microRNA biomarkers of pancreatic cancer using one-pot endonucleolytically exponentiated rolling circle amplification by CRISPR-Cas12a. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3794.

Target-promoted specific activation of m6A-DNAzyme for SPEXPAR-amplified and highly sensitive non-label electrochemical assay of FTO demethylase.

The demethylase of fat mass and obesity related protein (FTO) is critical to regulate the dynamic N6-methyladenosine (m6A) modification of eukaryotic mRNAs, and its overexpression has found to be closely related to the initiation of several cancers. On the basis of a target-promoted specific activation of DNAzyme strategy coupled with self-primer exponential amplification reaction (SPEXPAR) cycles and DNA supersandwich assemblies, the highly sensitive and label-free electrochemical FTO assay approach is established. The modification of the catalytic core nucleobase of the DNAzyme probe by m6A can inhibit its cleavage activity. The presence of target FTO catalyzes the elimination of the methyl group to restore the DNAzyme activity, which cleaves the hairpin substrates to trigger the SPEXPAR for yielding many ssDNAs. The capture of these DNAs on the sensor electrode leads to the initiation of supersandwich assembly formation of long dsDNAs. Tremendous electrochemical signal probe of [Ru(NH3)6]Cl3 are then absorbed on these dsDNAs to produce highly amplified catalytic currents with the assistance of K3[Fe(CN)6] for detecting trace FTO with 63.1fM detection limit. Furthermore, the sensor can be employed for selective assay of FTO in cell lysates, revealing the great potential of this sensing strategy for biomedical and biological study applications.

Label-free and sensitive fluorescent sensing of ten-eleven translocation enzyme via cascaded recycling signal amplifications

The sensitive detection of ten-eleven translocation (TET) dioxygenase is of significance for understanding the demethylation mechanism of 5-methylocytosine (5mC), which is responsible for a wide range of biological functions that can affect gene expression in eukaryotic species. Here, a non-label and sensitive fluorescence biosensing method for TET assay using TET1 as the model target molecule is established on the basis of target-triggered Mg2+-dependent DNAzyme and catalytic hairpin assembly (CHA)-mediated multiple signal amplification cascades. 5mC sites in the hairpin DNA probe are first oxidized by TET1 into 5-carboxycytosine, which are further reduced by pyridine borane into dihydrouracil, followed by its recognition and cleavage by the USER enzyme to liberate active DNAzyme and G-quadruplex sequences from the probe. The DNAzyme further cyclically cleaves the substrate hairpins to trigger subsequent CHA reaction and DNAzyme cleavage cycles for yielding many G-quadruplex strands. Thioflavin T dye then intercalates into G-quadruplexes to cause a magnificent increase of fluorescence for high sensitivity assay of TET1 with 47 fM detection limit. And, application of this method for TET1 monitoring in diluted serum has also been confirmed.

Molecularly imprinted polymer enhances affinity and stability over conventional aptasensor for blood clotting biomarker detection on regimented carbon nanohorn and gold nanourchin hybrid layers

This study aims to develop a rapid and sensitive biomimetic biosensor for the early detection of bleeding disorders by sensing human blood clotting factor IX protein (FIX). Authors used a dual sensitive detection strategy to identify FIX deficiency in human plasma, by incorporating aptamer recognition mechanism with molecular imprinted polymer (MIP) technology. Amino-modified truncated 2′ fluoro-RNA aptamer was used as macro-monomer in MIP synthesis. Produced MIP nanoparticles were immobilized through carbodiimide on Archimedean spiral interdigitated electrode surface for the electrical detection. The sensitivity was enhanced by surface modification using carbon nanohorn and gold nanourchin. Incorporating these hybrid materials and MIP, the enhanced FIX sensor sensitivity was at 6.06E-6 µA fM−1 µm−2. It was found that MIP incorporated sensor has 3-folds enhanced sensitivity with 40 fM detection limit compared to the conventional aptasensor. Validations have been given by sensing FIX level from human plasma serum with 10.7 pM sensitivity and by enzyme-linked apta-sorbent assay at 8 pM as a sensitivity. Moreover, the specificity of sensor also has proven with discrimination of the analogues. This research produced a novel biosensor for FIX detection and justifies that aptamer embedded MIP plays a crucial role for high-performance protein recognition in bleeding diathesis.

Ultrasensitive Electrochemical Detection of Mutated Viral RNAs with Single-Nucleotide Resolution Using a Nanoporous Electrode Array (NPEA).

The detection of nucleic acids and their mutation derivatives is vital for biomedical science and applications. Although many nucleic acid biosensors have been developed, they often require pretreatment processes, such as target amplification and tagging probes to nucleic acids. Moreover, current biosensors typically cannot detect sequence-specific mutations in the targeted nucleic acids. To address the above problems, herein, we developed an electrochemical nanobiosensing system using a phenomenon comprising metal ion intercalation into the targeted mismatched double-stranded nucleic acids and a homogeneous Au nanoporous electrode array (Au NPEA) to obtain (i) sensitive detection of viral RNA without conventional tagging and amplifying processes, (ii) determination of viral mutation occurrence in a simple detection manner, and (iii) multiplexed detection of several RNA targets simultaneously. As a proof-of-concept demonstration, a SARS-CoV-2 viral RNA and its mutation derivative were used in this study. Our developed nanobiosensor exhibited highly sensitive detection of SARS-CoV-2 RNA (∼1 fM detection limit) without tagging and amplifying steps. In addition, a single point mutation of SARS-CoV-2 RNA was detected in a one-step analysis. Furthermore, multiplexed detection of several SARS-CoV-2 RNAs was successfully demonstrated using a single chip with four combinatorial NPEAs generated by a 3D printing technique. Collectively, our developed nanobiosensor provides a promising platform technology capable of detecting various nucleic acids and their mutation derivatives in highly sensitive, simple, and time-effective manners for point-of-care biosensing.

Highly sensitive and label-free detection of DILI microRNA biomarker via target recycling and primer exchange reaction amplifications

MicroRNAs (miRNA) are closely associated with the development of diseases in life processes, and the sensitive detection of microRNA biomarkers is beneficial to disease diagnosis and treatment at early stage. Herein, by coupling the target sequence recycling with the primer exchange reaction (PER), a highly sensitive and non-label approach is constructed to detect miRNA-122, the biomarker for drug-induced liver injury. The target sequence cyclically displaces two hairpins on the DNA track via toehold-mediated strand displacement reactions with the assistance of the fuel DNA. The released hairpins further bind the primer to trigger the polymerase-aided PER process for the yield of plenty of G-quadruplex sequences, which then combine with the thioflavin T to drastically enhance its fluorescence for sensing miRNA-122 with a low 49.4 fM detection limit. Highly specific discrimination of the target miRNA-122 can also be realized with the proposed method. Because of the non-label format, high selectivity and sensitivity, such a method can be a convenient and universal means for sensing various biomarkers for disease diagnosis at the early stages.

Light‐Induced Solid‐State Protrusion of Gold Nanowires and Their Derivatives for Sensing Applications

AbstractGold nanowires (AuNWs) have been one of the key components for nanoelectronics and optoelectronic devices but the fabrication has been poorly customized for this purpose even though diverse synthetic strategies bloomed over the last decades. Here, the concept of light‐induced protrusion of AuNWs directly from solid state substrate is demonstrated, which is highly compatible for on‐chip fabrication of functional nanodevices. This is realized by irradiating the Au ion‐doped titanium dioxide (TiO2) films with continuous wave laser. The blue laser triggers the reduction and nucleation of Au(0) which grows vertically into AuNW bundles with diffusion‐controlled supply of Au ions within the TiO2 matrix. Thus, the morphology of the AuNWs can be tuned with irradiation time/power as well as the ion concentration, which can derive into Au nanorings (NRs) and NR stacks in 1D photonic crystal with reduced plasmon linewidth. Such light‐induced protrusion can also generate nanopillars by introducing Au seeds before irradiation. The surface‐enhanced Raman scattering performance of the AuNW bundles is excellent with fm detection limit. Such laser‐directed solid‐state growth technique is facile, versatile, cost‐effective site‐selective, ligand‐free, and wafer‐scalable, which opens up many promising applications in (bio)chemical sensing, photovoltaics, photocatalysis, plasmonic displays, and integrated nanoelectronic/optoelectronic devices.

Highly Sensitive Detection of Elevated Exosomal miR-122 Levels in Radiation Injury and Hepatic Inflammation Using an Aptamer-Functionalized SERS-Sandwich Assay.

Radiation-induced organ injury is one of the major fallouts noticed during radiotherapy treatment of malignancies and other detrimental radiation exposures. MicroRNA (miRNA), which is involved in multiple critical cellular processes, is released from the cells of damaged organs in cellular vesicles, commonly known as exosomes. Specifically, exosomal miR-122 is reported to be actively involved in radiation-actuated rectal and hepatic injuries or inflammation. In this work, we developed a surface-enhanced Raman spectroscopy (SERS) assay for the quantitative and targeted detection of exosomal miR-122 in mice after drug/radiation treatments. In particular, an aptamer-functionalized magnetic capturing element and Au shell nanoparticle (NP)-based SERS tags were utilized, which upon recognition of the target miRNA constituted a "sandwich" formation, with which an 8 fM limit of detection (LOD) could be achieved. Using this SERS assay, we further found that radiation injury led to the elevated expression of exosomal miR-122 in mice at 4 h postirradiation, confirmed by the quantitative real-time PCR method. It was demonstrated that the drug-induced hepatic inflammation could also be assessed via detecting miR-122 using this SERS method. As such, this work has demonstrated the achievement of a highly selective and sensitive probe of exosomal miRNA, which may thus open a gateway for promising usage in drug/radiation-induced inflammation.

Study on the Physical Properties of a SiNW Biosensor to the Sensitivity of DNA Detection.

SiNW (silicon nanowire) arrays consisting of 5- and 10-wires were fabricated by using an atomic force microscope—the local anodic oxidation (AFM-LAO) technique followed by wet chemical etching. Tetramethylammonium hydroxide (TMAH) and isopropyl alcohol (IPA) at various concentrations were used to etch SiNWs. The SiNWs produced were differed in dimension and surface roughness. The SiNWs were functionalized and used for the detection of deoxyribonucleic acid (DNA) dengue (DEN-1). SiNW-based biosensors show sensitive detection of dengue DNA due to certain factors. The physical properties of SiNWs, such as the number of wires, the dimensions of wires, and surface roughness, were found to influence the sensitivity of the biosensor device. The SiNW biosensor device with 10 wires, a larger surface-to-volume ratio, and a rough surface is the most sensitive device, with a 1.93 fM limit of detection (LOD).

Coenzyme−catalyzed electroinitiated reversible addition fragmentation chain transfer polymerization for ultrasensitive electrochemical DNA detection

Ultrasensitive detection of biomarkers at an early stage is generally limited by external influence factors such as high reaction temperature, complex operations, and sophisticated instruments. Here, we circumvent these problems by using nicotinamide adenine dinucleotide (NAD+) to control electroinitiated reversible addition fragmentation chain transfer (electro−RAFT) polymerization for biosensing that enables the detection of a few molecules of target DNA. In this coenzyme−catalyzed electro−RAFT polymerization, numerous ferrocenylmethyl methacrylate (FCMMA) as monomer with electrochemistry signal were linked to the biomarker on Au electrode. Afterwards, a strong oxidation peak appears at the potential of about 0.3 V that represents a typical oxidation potential of FCMMA. The sensitivity of this methodology was presented by detecting DNA from 10−1 to 104 fM concentration and detection limit (LOD) being down to 4.39 aM in 10 μL samples. This is lower by factors than detection limits of most other ultra−sensitive electrochemical DNA assays.

Dual amplification in a fluorometric acetamiprid assay by using an aptamer, G-quadruplex/hemin DNAzyme, and graphene quantum dots functionalized with D-penicillamine and histidine.

D-penicillamine and histidine-functionalized graphene quantum dot (DPA-GQD-His) was synthesized and applied in a fluorometric method for determination of acetamiprid using a G-quadruplex DNAzyme. At first DNA probe (probe 1) consists of a target-specific aptamer with two arms of DNA segments. Probe 1 was hybridized with DNA probe 2 composed of a single DNA sequence with two split G-rich DNA sequences. This leads to the formation of a triplex-to-G-quadruplex (TPGQ). Next, acetamiprid was hybridized with the aptamer in the TPGQ to release free DNA probe 2. The released probe 2, in the presence of of K+, undergoes a structural change into a stem-loop structure (by self-complementary hybridization and Hoogsteen hydrogen bonding) that bears a G-quadruplex structure. This is followed by conjugation with hemin to form the G-quadruplex/hemin DNAzyme. The DNAzyme catalyzes the oxidation of o-phenylenediamine by H2O2 to produce a yellow fluorescent product with excitation/emission maxima at 420/560nm. The oxidation product interacts with DPA-GQD-His to achieve a rapid energy transfer between DPA-GQD-His and oxidation product. This increases the fluorescence of the oxidation product and quenches the fluorescence of DPA-GQD-His. DPA-GQD-His also improves the catalytic activity of DNAzyme towards oxidation of ophenylenediamine oxidization and enhances fluorometric response to acetamiprid. The assay works in the 1.0 fM to 1.0nM acetamiprid concentration range and has a 0.38 fM detection limit. It was successfully applied to the determination of acetamiprid in tea. Graphical abstractThe study reported one double amplification strategy for ultrasensitive fluorescence detection of acetamiprid in tea with D-penicillamine and histidine-functionalized graphene quantum dots and G-quadruplex/heminDNAzyme. The analtyical method exhibits ultra high sensitivity, selectivity and rapidity of fluorescenceresponse to acetamiprid.

Sensitive colorimetric determination of microRNA let-7a through rolling circle amplification and a peroxidase-mimickingsystem composed of trimeric G-triplex and hemin DNAzyme.

The authors have incidentally found that the three tandem repeats of a 13-mer G-rich oligomer (with sequence 5'-TGG GAA GGG AGG G-3'; referred to as G3) can directly fold into a stable G3 trimer. The G3 trimer/hemin DNAzyme exhibits an about 3-foldhigher peroxidase-mimicking activity compared to the conventional G3/hemin DNAzyme. Combining this finding with rolling circle amplification (RCA), a colorimetric assay was developed for sensitive and specific determination of microRNA. In this method, each cycle of RCA generates three catalytic units. This leads to a significant signal amplification of the RCA. Using let-7a as a model analyte, the colorimetric method (best performed at 420nm) exhibits high sensitivity toward microRNA-let-7a with a 37 fM detection limit and an analytical range that covers 3 orders of magnitude. The method was applied to the determination of let-7a in some cell lysates. Graphical abstractThis G-triplex trimer-based rolling circle amplification (RCA) method can produce three catalytic units per RCA cycle, which can significantly improve the amplification efficiency of RCA.

Highly Sensitive Colorimetric Detection of a Variety of Analytes via the Tyndall Effect

This work initially reports the use of a quite familiar optical phenomenon of colloidal solutions, namely, the Tyndall Effect (TE) as signal readout for highly sensitive colorimetric chemical and biological analysis. Taking gold nanoparticles (GNPs) as a model colloid, the TE-inspired assay (TEA) is developed based on the conversion of a specific recognition event (e.g., the aptamer-analyte binding) into the aggregation of GNPs, leading to a significant TE enhancement. In the TEA, a cheap laser pointer pen is used as a hand-held light source, while a smartphone serves as a portable quantitative reader. The results show that the TE signaling strategy achieves a ∼1000-fold sensitivity improvement compared with the most common surface plasmon resonance signaling method using GNPs. The utility of the TEA is well demonstrated with the inexpensive, rapid, and portable detection of trace levels of analytes ranging from an important small-molecule drug (cocaine, ∼1.5 pM detection limit) to a protein biomarker (interferon-γ, ∼2.2 fM detection limit) and a toxic metal ion (Ag+, ∼1.4 nM detection limit). In addition, as the TE enhancement simply stems from the aggregation of either bare (unmodified) or modified GNPs, the TEA is universally applicable to almost all of the existing GNP-based liquid-phase colorimetric assays. The TEA method developed herein lights a new way for equipment-free point-of-care analysis in various fields including medical diagnosis, food safety evaluation, and environmental monitoring, especially in the resource-poor areas of the world.

Surface plasmon coupling electrochemiluminescence assay based on the use of AuNP@C3N4QD@mSiO2 for the determination of theShiga toxin-producing Escherichia coli (STEC) gene.

This work describes a surface plasmon coupling electrochemiluminescence (SPC-ECL) method for the determination of the Shiga toxin-producing Escherichia coli (STEC) gene. Firstly, gold nanoparticles (Au NPs) were encapsulated into a solid silica core (AuNP@SiO2). Secondly, graphite phase carbon nitride quantum dots (g-C3N4 QDs) were embedded in the mesoporous silica shell (mSiO2) to form nanospheres of type AuNP@C3N4QD@mSiO2. It is found that the surface plasmon coupling effect of the Au NPs in the solid silica core strongly enhances the ECL of the g-C3N4/K2S2O8 system. The mSiO2 carry much of the ECL luminophore (g-C3N4 QDs), and the co-reactant can readily pass the mesopores to react with QDs to give an ECL reaction. Because of these two features, the ECL is 3.8 times stronger compared to ECL sensing using g-C3N4 QDs only. Finally, AuNP@C3N4QD@mSiO2 was linked to the probe DNA to construct a competitive DNA sensor. When no target DNA is added, most of the capture DNA on the electrode is complementary to the probe DNA of AuNP@C3N4QD@mSiO2-probe DNA. At this time, the ECL signal is the strongest. When the target DNA is added, some of the capture DNA is paired with it and the remaining capture DNA is paired with the probe DNA. Consequently, less luminophore reaches the electrode and the signal is weaker. The method works in the 0.1 pM to 1nM concentration range and has a 9 fM detection limit. It was successfully applied to the ultrasensitive determination of the STEC gene in human serum. Graphical abstract Schematic illustration for the "egg-yolk puff" structured ECL sensor based on Au NPs, g-C3N4 QDs, and mesoporous silica shell.

A CuO-CeO2 composite prepared by calcination of a bimetallic metal-organic framework for use in an enzyme-free electrochemical inhibition assay for malathion.

An enzyme-free electrochemical method is described for the determination of trace levels of malathion. It is based on a nanostructured copper-cerium oxide (CuO-CeO2) composite prepared by calcination of a Cu(II)/Ce(III) metal-organic framework. The morphology, crystal structure and elemental composition of composite was studied by scanning electron microscopy, energy dispersive X-ray spectroscopy, X-ray diffraction and X-ray photoelectron spectroscopy. The principle for malathion determination is based on the fact that the redox signal of CuO (best measured at around -0.1V vs. SCE) (at 100mV/s) is inhibited by malathion due to affinity between CuO and the sulfur groups of malathion. The introduction of CeO2 into the composite system further improves the analytical performance. This is attributed to the unique microstructure and the synergistic effect between CuO and CeO2. Experimental parameters like solution pH value, Cu/Ce molar ratio, accumulation potential, accumulation time, and CuO-CeO2 volume on the electrode were optimized. The assay has a linear range of 10 fM to 100nM and a 3.3 fM detection limit (at S/N= 3). The electrode is selectively inhibited by malathion even in the presence of potentially interfering substances. Graphical abstract A sensitive and effective enzyme-free electrochemical sensor has been developed for the detection of malathion based on CuO-CeO2 composite derived from bimetallic metal-organic frameworks.

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.