Ultrasensitive Detection Method Research Articles

Porous Co3O4 nanodisks as robust peroxidase mimetics in an ultrasensitive colorimetric sensor for the rapid detection of multiple heavy metal residues in environmental water samples

The current method for rapid and ultrasensitive detection of multiple heavy metals in environmental water still face challenge. Herein, the porous Co3O4 nanodisks with robust peroxidase-mimicking activity were prepared, and its catalytic activity can be significantly inhibited by the heavy metals like Cd(II), Hg(II), Pb(II) and As, which makes us to establish an ultrasensitive and rapid colorimetric sensor for the detection of multiple heavy metals. Further investigation reveals the anticompetitive inhibition effect of heavy metals on peroxidase-mimicking activity. The colorimetric sensor displays excellent sensitivity and selectivity, and the limits of detection (LOD) for Cd(II), Hg(II), Pb(II) and As are 0.085 μg·L−1, 0.19 μg·L−1, 0.2 μg·L−1 and 0.156 μg·L−1, respectively. Notably, the absorbance variation will be greater than 0.5 as the concentration of heavy metals exceeds 5 μg·L−1, which can be clearly discriminated by the naked eyes. Moreover, the average recovery range of heavy metals in actual water samples is from 86.9% to 98.3%. The above results indicate that the proposed sensor exhibits excellent practical applicability for the rapid and ultrasensitive detection of multiple harmful heavy metals in several environmental water samples, which has potential bright application in protecting the environment and human health.

Plasmonic Biosensors for Single-Molecule Biomedical Analysis.

The rapid spread of epidemic diseases (i.e., coronavirus disease 2019 (COVID-19)) has contributed to focus global attention on the diagnosis of medical conditions by ultrasensitive detection methods. To overcome this challenge, increasing efforts have been driven towards the development of single-molecule analytical platforms. In this context, recent progress in plasmonic biosensing has enabled the design of novel detection strategies capable of targeting individual molecules while evaluating their binding affinity and biological interactions. This review compiles the latest advances in plasmonic technologies for monitoring clinically relevant biomarkers at the single-molecule level. Functional applications are discussed according to plasmonic sensing modes based on either nanoapertures or nanoparticle approaches. A special focus was devoted to new analytical developments involving a wide variety of analytes (e.g., proteins, living cells, nucleic acids and viruses). The utility of plasmonic-based single-molecule analysis for personalized medicine, considering technological limitations and future prospects, is also overviewed.

Rapid and sensitive label-free SERS determination of fucoxanthin in algae using gold nanoparticles

In this paper, we explored a rapid and ultra-sensitive detection method of fucoxanthin in algae by surface-enhanced Raman scattering. Gold nanoparticles (Au NPs) were prepared by the reduction of chloroauric acid with citric acid. An effective Raman indicator molecule, Rhodamine 6G(R6G) dye molecule, was used to characterize Au NPs that were used in SERS analysis to detect fucoxanthin. It was found that the detection limit of fucoxanthin could reach 10−6 mol·L−1. The characteristic peak of fucoxanthin at a wave number of 1520 cm−1was selected as the working curve, and it presents good linearity of fucoxanthin concentration with R2 = 0.932 in the range from 5×10−4∼1×10−7 M. We first proposed the rapid detection of fucoxanthin by surface-enhanced Raman scattering, which took only one minute to detect. In addition, the Raman strength of the pure algae or kelp and the addition of fucoxanthin was compared under the gold nanoparticle substrate, so as to verify the application value of SERS technology in the rapid determination of fucoxanthin.

Fe3O4@polydopamine and Exo III-assisted homogeneous biorecognition reaction for convenient and ultrasensitive detection of kanamycin antibiotic.

Herein, we report a Fe3O4@polydopamine (PDA) nanocomposite and exonuclease III (Exo III)-assisted homogeneous fluorescence biosensing method for ultrasensitive detection of kanamycin (Kana) antibiotic. A hairpin DNA containing the Kana-aptamer sequence (HP) was first designed for the highly specific biorecognition of the target analyte. Because of the aptamer biorecognition-induced structural change of HP and the highly effective catalyzed reaction of Exo III, a large amount of fluorophore labels were released from the designed fluorescence DNA probe. During the homogeneous reaction process, the Exo III-assisted dual recycling significantly amplified the fluorescence signal output. Moreover, the excessive probes were easily adsorbed and separated by the Fe3O4@PDA nanocomposite, which decreased the background signal and increased the signal-to-noise ratio. These strategies result in the excellent analytical performance of the method, including a very low detection limit of 0.023 pg mL-1 and a very wide linear range of six orders of magnitude. In addition, this method has convenient operation, excellent selectivity, repeatability and satisfactory reliability, and does not involve the design and utilization of complicated DNA sequences. Thus, it exhibits a promising prospect for practical applications.

Recent Advances in Aptamer-Based Biosensors for Detection of Pseudomonas aeruginosa.

Increasing concerns about nosocomial infection, food and environmental safety have prompted the development of rapid, accurate, specific and ultrasensitive methods for the early detection of critical pathogens. Pseudomonas aeruginosa is one of the most common pathogens that cause infection. It is ubiquitous in nature, being found in water, soil, and food, and poses a great threat to public health. The conventional detection technologies are either time consuming or readily produce false positive/negative results, which makes them unsuitable for early diagnosis and spot detection of P. aeruginosa. To circumvent these drawbacks, many efforts have been made to develop biosensors using aptamers as bio-recognition elements. Various aptamer-based biosensors for clinical diagnostics, food, and environmental monitoring of P. aeruginosa have been developed in recent years. In this review, we focus on the latest advances in aptamer-based biosensors for detection of P. aeruginosa. Representative biosensors are outlined according to their sensing mechanisms, which include optical, electrochemical and other signal transduction methods. Possible future trends in aptamer biosensors for pathogen detection are also outlined.

High-resolution, ultrasensitive and quantitative DNA double-strand break labeling in eukaryotic cells using i-BLESS.

DNA double-strand breaks (DSBs) are implicated in various physiological processes, such as class-switch recombination or crossing-over during meiosis, but also present a threat to genome stability. Extensive evidence shows that DSBs are a primary source of chromosome translocations or deletions, making them a major cause of genomic instability, a driving force of many diseases of civilization, such as cancer. Therefore, there is a great need for a precise, sensitive, and universal method for DSB detection, to enable both the study of their mechanisms of formation and repair as well as to explore their therapeutic potential. We provide a detailed protocol for our recently developed ultrasensitive and genome-wide DSB detection method: immobilized direct in situ breaks labeling, enrichment on streptavidin and next-generation sequencing (i-BLESS), which relies on the encapsulation of cells in agarose beads and labeling breaks directly and specifically with biotinylated linkers. i-BLESS labels DSBs with single-nucleotide resolution, allows detection of ultrarare breaks, takes 5 d to complete, and can be applied to samples from any organism, as long as a sufficient amount of starting material can be obtained. We also describe how to combine i-BLESS with our qDSB-Seq approach to enable the measurement of absolute DSB frequencies per cell and their precise genomic coordinates at the same time. Such normalization using qDSB-Seq is especially useful for the evaluation of spontaneous DSB levels and the estimation of DNA damage induced rather uniformly in the genome (e.g., by irradiation or radiomimetic chemotherapeutics).

Label-free tri-luminophores electrochemiluminescence sensor for microRNAs detection based on three-way DNA junction structure

Abstract A label-free microRNAs (miRNAs) electrochemiluminescence sensor was developed based on the synergistically enhanced ECL signal using novel ECL nanomaterial assembled by tri-luminophores perylenetetracarboxylic acid (PTCA) graphite-like carbon nitride nanosheet (g-C3N4 NS) and graphene carbon quantum dots (GQDs). The ECL intensity of our proposed nanocomposites increased significantly, by taking advantages of the synergistic promotion ECL mechanism of PTCA g-C3N4 and GQDs. Meanwhile, a three-way junction (TWJ) structure was introduced into the sensor design, whose conformation can be specifically induced in the presence of target miRNA. Thus, the formation of TWJ structure can hinder the contact between the co-reactant Na2S2O8 in the solution phase and the luminescent substrate, which produce a quenched ECL signal. Our proposed ECL biosensor provided a highly sensitive detection of miRNA-21 with a detection limit of 96 aM ranging from 100 aM to 1 nM. Furthermore, this ECL sensing system was able to successfully distinguish the target RNA from its homologous family and presented a promising response in human real samples. With excellent properties, the proposed ECL biosensor provided an efficient and ultrasensitive method for miRNAs detection.

A rapid, simple and ultrasensitive spectrofluorimetric method for the direct detection of metformin in real samples based on a nanoquenching approach.

Metformin (MET), as an oral antidiabetic and antihyperglycemic agent, is widely used to treat type II diabetes mellitus. Because of its increasing consumption, developing a fast, simple, and selective method to determine its concentration in biological samples (serum and urine) and pharmaceutical formulations (tablets) is of great interest. In this study, we used a FRET-based fluorescent nanosensor (Tb-phen-AgNPs system) for sensitive detection of MET in tablet and serum samples. This method is based on the enhancing effect of MET on the emission intensity of the Tb-phen complex, which is quenched by AgNPs via energy transfer process (turn off-on mode). A good linear relationship between the MET concentration and enhanced emission intensity of the Tb-phen-AgNPs system was observed in the range of (0.75-3.7) × 10-6 M under optimum conditions. Limit of detection and limit of quantitation were calculated to be 0.43 × 10-6 M and 1.31 × 10-6 M, respectively. This method was successfully used to determine MET concentrations in pharmaceutical dosage form and in spiked serum sample. The obtained recoveries from pharmaceutical formulation and serum sample were in the range 86.75-98.97% and 85.10-100.96%, respectively. Collectively, our results indicated that the method described here is simple, sensitive, cost effective, and free from interference. Therefore, it can be used as an effective and routine method for the direct and rapid determination of MET levels in biological samples such as serum.

Liquid Biomarkers for Pediatric Brain Tumors: Biological Features, Advantages and Perspectives.

Tumors of the central nervous system are the most frequent solid tumor type and the major cause for cancer-related mortality in children and adolescents. These tumors are biologically highly heterogeneous and comprise various different entities. Molecular diagnostics are already well-established for pediatric brain tumors and have facilitated a more accurate patient stratification. The availability of targeted, biomarker-driven therapies has increased the necessity of longitudinal monitoring of molecular alterations within tumors for precision medicine-guided therapy. Nevertheless, diagnosis is still primarily based on analyses of the primary tumor and follow-up is usually performed by imaging techniques which lack important information on tumor biology possibly changing the course of the disease. To overcome this shortage of longitudinal information, liquid biopsy has emerged as a promising diagnostic tool representing a less-invasive source of biomarkers for tumor monitoring and therapeutic decision making. Novel ultrasensitive methods for detection of allele variants, genetic alterations with low abundance, have been developed and are promising tools for establishing and integrating liquid biopsy techniques into clinical routine. Pediatric brain tumors harbor multiple molecular alterations with the potential to be used as liquid biomarkers. Consequently, studies have already investigated different types of biomarker in diverse entities of pediatric brain tumors. However, there are still certain pitfalls until liquid biomarkers can be unleashed and implemented into routine clinical care. Within this review, we summarize current knowledge on liquid biopsy markers and technologies in pediatric brain tumors, their advantages and drawbacks, as well as future potential biomarkers and perspectives with respect to clinical implementation in patient care.

A highly sensitive, fast responsive and reversible naphthalimide-based fluorescent probe for hypochlorous acid and ascorbic acid in aqueous solution and living cells.

It is very important to exploit real-time, ultrasensitive and specific visualization detection methods for hypochlorous acid/hypochlorite (HOCl/ClO-) in biological systems as they are the guardians of the human immune system against pathogens invasion. In our work, we designed a novel reversible naphthalimide-based fluorescent probe NAP-OH to recognize HClO/ClO- with a unique selective colorimetric and fluorescent response, a short response time (<8 s) and a high sensitivity (10.3nM). In addition, NAP-OH exhibits a novel on-off-on fluorescence response to ClO-/ascorbic acid (AA) with good cycle stability. The fluorescence signal is quenched because HClO/ClO- oxidizes the subunit of NAP-OH to the segment 2,2,6,6-tetramethyl-1-oxo-piperidinium in NAP-O, which can be reduced by AA with the recovery of fluorescence. Finally, the confocal fluorescence imaging has been performed, which proves that NAP-OH can satisfactorily monitor intracellular endogenous and exogenous HClO/AA redox cycles.

Static interaction between colloidal carbon nano-dots and aniline: A novel platform for ultrasensitive detection of aniline in aqueous media

Aniline has been classified as a probable human carcinogen which can cause severe respiratory tract irritation and congestion to humans. Here, we propose using ultra-small (1.8 nm) colloidal nitrogen-doped carbon nano-dots (N-CDs), that is green synthesized at a low temperature of only 100 °C from ultrasonic-processed water-soluble folic acid, as a novel turn-off fluorescent chemo-sensor for ultrasensitive detection of aniline in aqueous medium. It was found that the Stern-Volmer relation describes very well the fluorescence quenching of N-CDs by aniline and confirms a static quenching mechanism between them. Compared to other techniques, our N-CDs based optical detection method, is the simplest and the most ultrasensitive method for aniline liquid detection, reaching an exceptional low detection limit of 3.57 nM (0.332 ppb). The proposed method was successfully applied for determination of aniline in a tap water sample without significant interferences from the matrix.

Single-electron detection utilizing coupled nonlinear microresonators

Since the discovery of the electron, the accurate detection of electrical charges has been a dream of the scientific community. Owing to some remarkable advantages, micro/nanoelectromechanical system-based resonators have been used to design electrometers with excellent sensitivity and resolution. Here, we demonstrate a novel ultrasensitive charge detection method utilizing nonlinear coupling in two micromechanical resonators. We achieve single-electron charge detection with a high resolution up to 0.197 ± 0.056 {mathrm{e}}/sqrt {{mathrm{Hz}}} at room temperature. Our findings provide a simple strategy for measuring electron charges with extreme accuracy.

Simplified Digital Enzyme-Linked Immunosorbent Assay Using Tyramide Signal Amplification and Fibrin Hydrogels.

Many protein biomarkers occur at very low concentrations in biofluids like blood and saliva, and ultrasensitive detection methods are required in order to measure them. Approaches such as digital enzyme-linked immunosorbent assays (ELISA) and single molecule arrays (Simoa) have been developed to accurately quantitate protein concentrations as low as attomolar levels. Although these techniques are being implemented in research and clinical laboratories to develop ultrasensitive clinical diagnostic assays, the size and cost of the instruments required to run these digital assays have precluded them from being implemented into point-of-care diagnostic formats. Here, we report the development of a simplified digital ELISA format that is more amenable to point-of-care technologies, referred to as catalyzed reporter deposition digital ELISA (CARD-dELISA). On-bead signal generation using the CARD tyramide signal amplification technique is combined with bead immobilization in fibrin hydrogels for single molecule counting in a simplified workflow format. CARD-dELISA allows for ultrasensitive protein detection (IL-6: ∼1 fM) with a dynamic range similar to the conventional Simoa assay. We use CARD-dELISA to measure IL-6 in saliva samples and show good agreement with conventional Simoa.

UMEPPI: An ultrasensitive detection method for protein-protein interaction.

UMEPPI: An ultrasensitive detection method for protein-protein interaction.

Experimental exploration of dynamic phase transitions and associated metamagnetic fluctuations for materials with different Curie temperatures.

We study dynamic magnetic behavior in the vicinity of the dynamic phase transition (DPT) for a suitable series of samples that have different Curie temperatures T_{C}, which thus enables us to experimentally explore the role of the reduced temperature T/T_{C} in the DPT. For this purpose, we fabricate Co_{1-x}Ru_{x} epitaxial thin films with uniaxial in-plane anisotropy by means of sputter deposition in the concentration range 0.0≤x≤0.26. All samples are ferromagnetic at room temperature, exhibit an abrupt magnetization reversal along their easy axis, and represent a unique T_{C} and thus T/T_{C} ratio according to their Ru concentration. The dynamic magnetic behavior is measured by using an ultrasensitive transverse magneto-optical detection method and the resulting dynamic states are explored as a function of the applied magnetic field amplitude H_{0} and period P, as well as an additional bias field H_{b}, which is the conjugate field of the dynamic order parameter Q. Our experimental results demonstrate that the qualitative behavior of the dynamic phase diagram is independent of the T/T_{C} ratio and that for all T/T_{C} values we observe metamagnetic anomalies in the dynamic paramagnetic state, which do not exist in the corresponding thermodynamic phase diagram. However, quantitatively, these metamagnetic anomalies are very strongly dependent on the T/T_{C} ratio, leading to an about 20-fold increase of large metamagnetic fluctuations in the paramagnetic regime as the T/T_{C} ratio increases from 0.37 to 0.68. Also, the phase space range in which these anomalous metamagnetic fluctuations occur extends closer and closer to the critical point as T/T_{C} increases.

Ultrafast visual nucleic acid detection with CRISPR/Cas12a and rapid PCR in single capillary

Rapid and sensitive nucleic acid detection is critical for point-of-care testing (POCT). Here, we proposed an ultrasensitive visual nucleic acid detection method that could be accomplished in <10 min. By integrating rapid PCR amplification and Cas12a cleavage as one-pot reaction in capillary, the operation was greatly simplified and amplicon contamination was totally avoided. Rapid PCR amplification was performed in portable thermo cups and Cas12a cleavage reaction was triggered with body-heat. Fluorescent results could be easily identified by naked eye with the help of a mini UV-touch. Results indicated that the proposed method possesses detection sensitivity at single molecule level, with high robustness, specificity and reliability. Thus, we have created a field-deployable nucleic acid detection platform with high performance and minimal equipment. It has broad application prospects for POCT in fields ranging from food safety determination to clinical diagnosis.

Phytosynthesis of Palladium Nanoclusters: An Efficient Nanozyme for Ultrasensitive and Selective Detection of Reactive Oxygen Species.

Hydrogen peroxide is a low-reactivity reactive oxygen species (ROS); however, it can easily penetrate cell membranes and produce highly reactive hydroxyl radical species through Fenton’s reaction. Its presence in abnormal amounts can lead to serious diseases in humans. Although the development of a simple, ultrasensitive, and selective method for H2O2 detection is crucial, this remains a strategic challenge. The peroxidase mimetic activity of palladium nanoclusters (PdNCs) has not previously been evaluated. In this study, we developed an ultrasensitive and selective colorimetric detection method for H2O2 using PdNCs. An unprecedented eco-friendly, cost-effective, and facile biological method was developed for the synthesis of PdNCs. This is the first report of the biosynthesis of PdNCs. The synthesized nanoclusters had a significantly narrow size distribution profile and high stability. The nanoclusters were demonstrated to possess a peroxidase mimetic activity that could oxidize peroxidase substrate 3,3′,5,5′-tetramethylbenzidine (TMB). Various interfering substances in serum (100 μM phenylalanine, cysteine, tryptophan, arginine, glucose, urea, Na+, Fe2+, PO43−, Mn+2, Ca2+, Mg2+, Zn2+, NH4+, and K+) were included to evaluate the selectivity of the assay, and oxidation of TMB occurred only in the presence of H2O2. Therefore, PdNCs show an efficient nanozyme for the peroxidase mimetic activity. The assay produced a sufficient signal at the ultralow concentration of 0.0625 µM H2O2. This colorimetric assay provides a real-time, rapid, and easy-to-use platform for the detection of H2O2 for clinical purposes.

Early diagnosis with ultrasensitive ELISA.

Accurate, rapid and simple detection methods are required to facilitate early diagnosis of various disorders including infectious and lifestyle diseases as well as cancer. These detection approaches reduce the window of infection, i.e., the period between infection and reliable detection. Optimally, these methods should target protein as an indicator of pathogenic microbes as well as other biomarkers. For example, although nucleic acid is easily detected by polymerase chain reaction (PCR), these markers are also present in dead microbes, and, in the case of mRNA, it is not known whether this target was successfully translated. Accordingly, early diagnostic approaches require the development of ultrasensitive protein detection methods. In this chapter, we introduce an ultrasensitive enzyme-linked immunosorbent assay (ELISA) which combines a traditional sandwich-based immunoassay with thionicotinamide adenine dinucleotide (thio-NAD) cycling. The performance characteristics of this unique approach are reviewed as well as its potential role in providing a novel and ultrasensitive diagnostic tool in the clinical laboratory.

Clinical implication and usefulness of de novo EGFR T790M mutation in lung adenocarcinoma with EGFR-tyrosine kinase inhibitor sensitizing mutation

ABSTRACT Detection rate of de novo EGFR T790 M mutation was increased up to 80% through recent ultrasensitive detection methods. Here, we investigated the clinical significance and its usefulness of detecting de novo EGFR T790 M using ultrasensitive droplet-digital polymerase chain reaction (ddPCR) method. In total, 102 cases diagnosed as lung adenocarcinoma with EGFR-tyrosine kinase inhibitor (TKI) sensitizing mutations (mEGFR) and had been treated with 1st ~ 2nd generation EGFR-TKI alone were enrolled for this study. De novo T790 M status was tested using the tissues at the initial diagnosis and positivity was defined as the ratio of T790 M/wild-type copies over 0.00294 by ddPCR. Seventy patients (68.6%) harbored the de novo T790 M. De novo T790 M was more frequently detected in cases with EGFR L858 R mutation than those with EGFR exon 19 deletion (E19d) mutations (P = 0.024). Forty-three patients underwent rebiopsy due to disease progression. The cases who experienced progression due to acquired T790 M were more likely to have E19d at initial diagnosis and the presence of de novo T790 M and the ratio of T790 M/wild-type copies did not relate to the emergence of acquired T790 M. On the other hand, the cases with a longer duration of disease-control by EGFR-TKI had higher change to get acquired T790 M mutation (P-value = 0.040). The presence of de novo T790 M has limitation in predicting disease progression by acquired T790 M, suggesting that identifying de novo T790 M through the ultrasensitive methods may not be necessary identifying patients who would be beneficial by 3rd-generation EGFR-TKI as the 1st line treatment.

High-Efficiency CNNS@NH2-MIL(Fe) Electrochemiluminescence Emitters Coupled with Ti3C2 Nanosheets as a Matrix for a Highly Sensitive Cardiac Troponin I Assay.

Synthesis of a highly efficient electrochemiluminescence (ECL) luminescent material is one of the effective means to improve the sensitivity of the sensor. In this study, an efficient ECL luminescent nanomaterial, carbon nitride nanosheet (CNNS) decorated amino-functional metal-organic frameworks (CNNS@NH2-MIL(Fe)) were synthesized for sensitive ECL detection of cardiac troponin I (cTn-I). The synthesized CNNS@NH2-MIL(Fe) realized the effective mass loading of CNNS, and more importantly, the NH2-MIL(Fe) could expedite the reduction of coreactant S2O82- to produce abundant ECL reaction intermediate SO4•- near CNNS, thus, shortening the distance between SO4•- and the excited state of CNNS with less energy loss to extremely enhance the ECL signal of CNNS. Furthermore, the ECL signal of the immunosensor could be further enhanced when the Ti3C2 nanosheet was used as the matrix to capture primary anti-cTn-I due to the reason that Ti3C2 not only exhibited a large surface area and excellent metallic conductivity, but also could act as a coreaction accelerator to speed up the reduction of S2O82- with plenty of SO4•- generated. Therefore, this proposed ECL immunosensor using CNNS@NH2-MIL(Fe) as a signal probe and Ti3C2 as a sensing matrix exhibited a significantly enhanced ECL signal and had a high sensitivity and excellent selectivity for cTn-I. Consequently, this multiple signal amplification strategy provided an effective method for trace protein ultrasensitive detection in ECL bioanalysis.