Ultrasensitive Detection Method Research Articles

Understanding the Role of Extracellular Vesicles in Lenalidomide-Resistance Multiple Myeloma

Multiple myeloma (MM) is a plasma cell malignancy that develops by the accumulation of various genetic abnormalities. In recent years, the prognosis of MM has improved by the development of novel drugs including immunomodulatory drugs (IMiDs), proteasome inhibitors, and antibody drugs. However, MM cells acquired drug resistance by long-term exposure to these therapeutic drugs. MM is a multiclonal disease, and various clone subtypes develop within the bone marrow microenvironment. It has been suggested that drug resistant phenotype could transmit from resistant clones to sensitive ones, and also to immune cells, or mesenchymal stem cells, resulting in the change of bone marrow microenvironment suitable for MM cell survival. It has been shown that extracellular vesicles (EVs) are one of the means of information transmission. EVs are secreted from almost all cells, and the amount of EV secretion is particular high from cancer cells. It was already known that cancer-derived EVs transmitted information associated with cancer progressions such as angiogenesis, metastasis, and drug resistance to the surrounding cells. Thus, EVs were proposed to play an important role in acquisition of drug resistance even though the mechanisms have not been fully understood in MM.In order to understand the mechanism of drug resistance in MM mediated by EVs, lenalidomide resistant cell lines were established by long-term exposure of lenalidomide. The amount of EVs was measured by ExoScreen, which is ultra-sensitive detection method of EVs by measuring surface protein of EVs, such as, CD9 and CD63, and by the nanoparticle tracking analysis. We found that lenalidomide resistant cell lines in KMS21R, KMS27R and KMS34R cell lines secreted about twice more EVs than their parental cell lines, and the amount of EV secretion was correlated with the drug sensitivity of lenalidomide. Suppression of EV secretion by knockdown of TSG101, which is known for EV secretion-associated protein, did not affect lenalidomide resistance. We could suppress the EV secretion to two-thirds, however cell proliferation and caspase activity were not change. From these results, we postulated the two possibilities; 1) EV secretion pathway other than TSG101 is associated with drug resistance via EVs; 2) EV derived from lenalidomide resistant cells can affect the cells exist in bone marrow microenvironment. From these hypotheses, we have done the following experiments. Firstly, to identify the genes which involved in EV secretion pathway associated with drug resistance, RNA sequence among the drug-resistant cell lines and their parental cell lines was performed. Drug resistant cell lines had some genetic abnormality, for instance immune system or angiogenesis. Now, we are detecting the EV secretion associated genes in drug resistant cell lines.Secondly, EV derived from the drug-resistant cell lines and EV derived from the parent cell lines were added to drug sensitive MM cell lines, then lenalidomide is added after 24hr. The cell proliferation and apoptosis assay were evaluated after 48hr. EV derived from the drug-resistant cell lines in KMS34R cell line significantly inhibited cell death measured by MTS assay and apoptosis assay compared with those from the drug sensitive KMS21 and KMS34 cell lines. EVs from KMS34R cell line, which is the most progressed cell line we established, could more transmit drug resistance than those from other cell lines. These results suggested that drug resistance was transmitted from drug-resistant cell lines to non-resistant cell lines via EVs.Now, we are analyzing the component of EV from drug-resistant MM cells by proteome analysis to identify the molecules associated with lenalidomide resistance in MM. In addition, we are investigating the molecules which associated with the secretion of EVs from drug-resistance MM. These results prompted us to hypothesize that attenuating the function of a molecule responsible for EV secretion could lead to the inhibition of cancer development such as drug resistance. It is expected that EVs will be novel therapeutic targets in refractory or relapsed MM. DisclosuresHattori:IDAC inc.: Research Funding; Takeda: Research Funding.

I-BLESS is an ultra-sensitive method for detection of DNA double-strand breaks

Maintenance of genome stability is a key issue for cell fate that could be compromised by chromosome deletions and translocations caused by DNA double-strand breaks (DSBs). Thus development of precise and sensitive tools for DSBs labeling is of great importance for understanding mechanisms of DSB formation, their sensing and repair. Until now there has been no high resolution and specific DSB detection technique that would be applicable to any cells regardless of their size. Here, we present i-BLESS, a universal method for direct genome-wide DNA double-strand break labeling in cells immobilized in agarose beads. i-BLESS has three key advantages: it is the only unbiased method applicable to yeast, achieves a sensitivity of one break at a given position in 100,000 cells, and eliminates background noise while still allowing for fixation of samples. The method allows detection of ultra-rare breaks such as those forming spontaneously at G-quadruplexes.

Detection of a Traumatic Brain Injury Biomarker at the 10fg/mL Level.

The detection of minute amounts of protein biomarkers in body fluids is believed to provide early diagnosis and prognosis of mild traumatic brain injury (mTBI). An ultrasensitive detection method was used to detect S100B, the most studied potential marker for the diagnosis of mTBI. The detection method was a modified electrochemical immunoassay technique that provides voltage controlled intrinsic current signal amplification. The sandwich immune complex of S100B was formed on the working electrode of the screen-printed electrode. The gating voltage provides amplification of the current signal that flows through the complex. S100B was spiked in human serum. The limit of detection of S100B in human serum was 10fg/mL. The calibration curves cover four orders of magnitudes from 10fg/mL to 10ng/mL. The specificity of the detection was demonstrated using TAU protein, which is another marker for mTBI. The results reported in this work using the field effect enzymatic detection (FEED)-based immunoassay indicate the feasibility of using this method for the detection of extremely low concentrations of markers of mTBI in human serum. This method can be developed as a platform for a range of markers of mTBI.

Analyte-triggered cyclic autocatalytic oxidation amplification combined with anupconversion nanoparticle probe for fluorometricdetection of copper(II).

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

Rapid detection of taste and odor compounds in water using the newly invented chemi-ionization technique coupled with time-of-flight mass spectrometry

Taste and odor (T&O) compounds are widespread in water environments and have attracted considerable public attention. Nowadays, the standard detections of these chemicals rely mainly on off-line methods such as GC-MS or evaluation by trained analysts' senses. In this study, we report a method for the rapid detection of T&O compounds in water by exploiting a newly invented chemi-ionization source, in combination with headspace vapor measurement at room temperature. The calibrated limits of detection (LODs) of 2-methylbutyraldehyde, methyl tert-butyl ether (MTBE), methyl methacrylate (MMA), 2-isobutyl-3-methyoxypyrazine (IBMP), and 2-isopropyl-3-methoxypyrazine (IPMP) are in the range of 3.5–50.2 ng L−1, and the estimated LODs of 2-methylisoborneol (2-MIB) and geosmin (GSM) are 0.25 and 0.77 ng L−1, respectively. The calibration results reveal that the instrumental LODs for 2-methylbutyraldehyde, MTBE, MMA, β-cyclocitral, 2-MIB, and GSM are 1–2 orders of magnitude better than the odor thresholds of humans. The accuracy, precision, recovery, and linearity (R2) of the method are tested. Water samples from city tap water and three rivers in Beijing are assessed using this technique, and the typical T&O compositions are observed with concentrations ranging from 0.2 to 297 ng L−1. The new ultra-sensitive rapid detection method shows comparable sensitivities to the existing off-line technique and displays great potential for real-time detection of T&O pollution in water environments.

Ultra-sensitive spectroscopy of OH radical in high-temperature transient reactions.

The hydroxyl (OH) radical is arguably the most important transient radical in high-temperature gas-phase combustion reactions, yet it is very difficult to measure because of its high reactivity and, thus, short lifetime and low concentration. This work reports the development of a novel method for ultra-sensitive, quantitative, and microsecond-resolved detection of OH based on UV frequency-modulation spectroscopy (FMS). To the best of the authors' knowledge, this is the first FMS demonstration in the near-UV spectral region for detection of short-lived radical species. Shot-noise-limited detection was achieved at an optical power of 25mW. A proof-of-concept experiment in a tabletop H2O/He microwave discharge cell has reached a 1σ minimum detectable absorbance (MDA) of less than 2×10-4 over 1MHz measurement bandwidth. High-temperature OH measurement was demonstrated in a 15cm diameter shock tube, where a typical MDA of 3.0×10-4 was achieved at 1330K, 0.38atm, and 1MHz. These preliminary results have outperformed the previous best MDA by more than a factor of 3; further improvement by another order of magnitude is anticipated, following the strategies outlined at the end of this Letter. The current method paves the path to parts per billion (ppb) -level OH detection capability and offers prospects to significantly advance fundamental combustion research by enabling direct observation of OH formation and scavenging kinetics during key stages of fuel oxidation that were inaccessible with previous methods.

Abstract 2613: Predictive impact of low-frequency pretreatment T790M mutation in patients with EGFR-mutated non-small cell lung cancer treated with EGFR tyrosine kinase inhibitors

Abstract Background: T790M mutation in EGFR accounts for nearly 50% of the acquired mechanism of resistance to EGFR-tyrosine kinase inhibitors (TKIs). Previous studies suggested that EGFR T790M mutation was also detected in a considerable number of EGFR-TKI-naïve non-small-cell lung cancer (NSCLC) patients utilizing ultrasensitive detection methods such as droplet digital PCR (ddPCR). Here we investigated the significance of low-frequency pretreatment EGFR T790M mutation (preT790M) and its association with cancer molecular heterogeneity in the efficacy of EGFR-TKIs. Materials and methods: Fifty-two advanced NSCLC patients harboring activating EGFR mutations treated with first-line EGFR-TKIs at Osaka City University Hospital between August 2013 and July 2016 were enrolled in the study. DNAs from tumor biopsies at diagnosis were available from 44 patients for detecting preT790M by the cobas® EGFR Mutation Test v2 (cobas) and ddPCR (RainDance Technologies) and those from 33 were available for assessing the actionable mutations in 50 genes by next-generation sequencing (NGS). NGS was performed on the Ion PGM using Ion AmpliSeq Cancer Hotspot Panel v2 (Life Technologies). Paired biopsies before EGFR-TKIs treatment and at disease progression (PD) were obtained from 15 patients to assess the T790M mutation by cobas. Results: The overall detection rate of preT790M by ddPCR was 40.9% (18/44), while not detected in any case by cobas. The median progression free survival (mPFS) was 10.0 months for the patients with preT790M and 13.5 months for those without preT790M (p=0.288), respectively. When divided into 3 categories based on the frequency of preT790M such as high with T790M allele frequency (AF) >0.3%, low with AF ≤0.3% and undetected, 12 patients with high AF had a relatively shorter mPFS than 6 with low AF (p=0.090) and 26 without detectable preT790M (p=0.046) (7.7 vs 17.1 vs 13.5 months, respectively). NGS revealed 18 additional coexisting actionable mutations in 15 out of 33 patients: TP53 (n=7), PIK3CA (n=5), CTNNB1 (n=3) and uncommon EGFR (n=3). There was a trend for patients with higher preT790M AF to harbor less additional coexisting mutations (27% with high AF, 60% with low AF and 53% without preT790M, p=0.320). In 15 paired biopsies, T790M mutation was detected in 60% (9/15) at PD by cobas in the clinical setting; 67% (2/3) with high AF, 50% (1/2) with low AF and 60% (6/10) without detectable preT790M, suggesting T790M mutation after first-line EGFR-TKIs developed from both clonal selection and secondary acquisition models. Conclusion: Results of our study indicated that EGFR-mutated NSCLCs with higher AF preT790M >0.3% had significantly shorter duration of response to EGFR-TKIs. In addition, the AF of preT790M in EGFR-mutated NSCLC may be associated with coexisting actionable mutation load potentially affecting the efficacy of EGFR-TKIs. Citation Format: Yoshiya Matsumoto, Kenji Sawa, Jun Oyanagi, Mitsuru Fukui, Naoki Yoshimoto, Tomohiro Suzumura, Shigeki Mitsuoka, Kazuhisa Asai, Tatsuo Kimura, Nobuyuki Yamamoto, Tomoya Kawaguchi, Kazuto Hirata, Yasuhiro Koh. Predictive impact of low-frequency pretreatment T790M mutation in patients with EGFR-mutated non-small cell lung cancer treated with EGFR tyrosine kinase inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2613.

CancerDetector: ultrasensitive and non-invasive cancer detection at the resolution of individual reads using cell-free DNA methylation sequencing data.

The detection of tumor-derived cell-free DNA in plasma is one of the most promising directions in cancer diagnosis. The major challenge in such an approach is how to identify the tiny amount of tumor DNAs out of total cell-free DNAs in blood. Here we propose an ultrasensitive cancer detection method, termed ‘CancerDetector’, using the DNA methylation profiles of cell-free DNAs. The key of our method is to probabilistically model the joint methylation states of multiple adjacent CpG sites on an individual sequencing read, in order to exploit the pervasive nature of DNA methylation for signal amplification. Therefore, CancerDetector can sensitively identify a trace amount of tumor cfDNAs in plasma, at the level of individual reads. We evaluated CancerDetector on the simulated data, and showed a high concordance of the predicted and true tumor fraction. Testing CancerDetector on real plasma data demonstrated its high sensitivity and specificity in detecting tumor cfDNAs. In addition, the predicted tumor fraction showed great consistency with tumor size and survival outcome. Note that all of those testing were performed on sequencing data at low to medium coverage (1× to 10×). Therefore, CancerDetector holds the great potential to detect cancer early and cost-effectively.

Ultrasensitive Detection of Formaldehyde in Gas and Solutions by a Catalyst Preplaced Sensor Based on a Pillar[5]arene Derivative

Abnormal formaldehyde (FA) is known to induce a variety of diseases. Herein, we report a novel and efficient method for ultrasensitive detection of formaldehyde in gas and solutions by a catalyst preplaced sensor based on the pillar[5]arene derivative (DP5J). By the catalyzation of (CF3SO3)2Bi, DP5J could selectively and sensitively sense formaldehyde through an aggregation-induced emission (AIE) “turn-on” response within 7.5 s, and the detection limits for formaldehyde is 3.27 × 10–9 M. Moreover, a FA test kit was prepared by loading the catalyst (CF3SO3)2Bi preplaced DP5J sensor (DP5J-Bi) on a silica gel plate. The test kit could conveniently and efficiently detect formaldehyde in gas or solution with ultrasensitivity. The catalysts preplaced method and FA reaction-induced AIE fluorescence “turn-on” mechanism is a novel approach to achieve ultrasensitive detection of FA. Importantly, it is also a novel approach for the efficient detection of other volatile organic compounds.

Rapid, On-Site, Ultrasensitive Melamine Quantitation Method for Protein Beverages Using Time-Resolved Fluorescence Detection Paper

To ensure protein beverage safety and prevent illegal melamine use to artificially increase protein content, a rapid, on-site, ultrasensitive detection method for melamine must be developed because melamine is detrimental to human health. Herein, an ultrasensitive time-resolved fluorescence detection paper (TFDP) was developed to detect melamine in protein beverages within 15 min using a one-step sample preparation. The lower limits of detection were 0.89, 0.94, and 1.05 ng/mL, and the linear ranges were 2.67-150, 2.82-150, and 3.15-150 ng/mL (R2 > 0.982) for peanut, walnut, and coconut beverages, respectively. The recovery rates were 85.86-110.60% with a coefficient of variation <7.80% in the spiking experiment. A high specificity was observed in the interferent experiment. When detecting real protein beverage samples, the TFDP and ultraperformance liquid chromatography-tandem mass spectrometer (UPLC-MS/MS) results were consistent. This method is a promising alternative for rapid, on-site detection of melamine in beverages.

A sensitive detection assay based on signal amplification technology for Alzheimer's disease's early biomarker in exosome

Alzheimer's disease (AD) considered as the third health “killer” has seriously threatened the health of the elderly. However, the modern diagnostic strategies of AD present several disadvantages: the low accuracy and specificity resulting in some false-negative diagnoses, and the poor sensitivity leading to a delayed treatment. In view of this situation, a enzyme-free and target-triggered signal amplification strategy, based on graphene oxide (GO) and entropy-driven strand displacement reaction (ESDR) principle, was proposed. In this strategy, when the hairpin structure probes (H)specially binds with beta-amyloid-(1–42) oligomers (Aβ42 oligomers), it's structure will be opened, causing the bases complementary to FAM-labeled replacement probes R (R1 and R2) exposed. At this time, R1 and R2 will hybridize with H, resulting in the bound Aβ42 oligomers released. The released Aβ42 oligomers would participate in the next cycle reaction, making the signal amplified. As a quencher, GO could absorb the free single-stranded DNA R1 and R2 and quench their fluorescence; however, the DNA duplex still exists free and keeps its signal-on. Through the detection of Aβ42 oligomers in exosomes, this ultrasensitive detection method with the advantages of low limit of detection (LOD, 20 pM), great accuracy, excellent precision and convenience provides an excellent prospect for AD's early diagnosis.

Enzyme-Free Immunoassay Using Silver Nanoparticles for Detection of Gliadin at Ultralow Concentrations

Determination of biomarkers in clinical or food samples is of crucial importance for monitoring, prevention, and care of public health. The standard procedure used for this purpose is the enzyme-linked immunosorbent assay (ELISA), which makes use of the specific antibody–antigen biorecognition and the catalytic effect of the enzymes. One of the main shortcomings of this technique is the use of enzymes that often present low chemical and thermal stabilities compared to other chemicals. Other drawbacks include the nonspecific binding process that could lead to false-positive results, the use of relatively large amounts of the sample, and the number of time-consuming steps involved. Recently, an enzyme-free and ultrasensitive analytical method for antigen detection denoted as intensity depletion immunolinked assay (IDILA) has been proposed by our laboratory. The assay is based on the inhibition to form Ag nanosphere dimers linked by a specific antibody in the presence of the corresponding antigen. In this work, we go a step further demonstrating how the performance of this method could be improved by using silver nanoparticles (Ag NPs) of different diameters (58 and 78 nm). The experiments are performed for detecting gliadin, an antigen of utmost importance in celiac disease, and the results are compared with ELISA, the standard technique homologated by the Food Codex Alimentarius. It is found that the IDILA assay could be around 1000 or 10 000 times more sensitive than ELISA, also having lower limits of detection, depending on the conditions explored (fraction of dimers and Ag NP diameter). Using the appropriate conditions, the IDILA assay is shown to be able to detect femtomolar concentrations of the antigen, besides being robust, reliable, cheap, rapid (around 2 h), and of easy implementation using the standard equipment and biomolecular reagents used for the ELISA assay.

A SERS-based multiple immuno-nanoprobe for ultrasensitive detection of neomycin and quinolone antibiotics via a lateral flow assay.

The authors describe an ultrasensitive method for simultaneous detection of neomycin (NEO) and quinolones antibiotics (QNS). It is based on the use of (a) two immuno-nanoprobes (a probe for NEO and a probe for QNS), (b) surface-enhanced Raman scattering (SERS) detection, and (c), a portable lateral flow assay (LFA). The two probes consist of gold nanoparticles (AuNPs) conjugated to the Raman active molecule 4-aminothiophenol (PATP), and to monoclonal antibody against NEO (NEO mAb) or against NOR (NOR mAb). Quantitative detection of NEO and QNS was realized via SERS of the PATP-coated AuNPs captured in the test line of a LFA. Under optimized condition, the visual limits of LFA are 10ng·mL-1 for NEO and 200ng·mL-1 for NOR, and with LODs down to 0.37pg·mL-1 and 0.55pg·mL-1 by using SERS. The NEO test line is not interfered by the NEO analogues gentamycin, streptomycin and tobramycin, but the NOR test line suffers from different degrees of cross-reactivity (CR) to 12 common other QNS, the CRs ranging from 1.5% to 136%. The recoveries of NEO and NOR from spiked milk samples ranged between 86% and 121%, with relative standard deviations (RSD) from 3% to 6%. The method is highly sensitive, accurate and effective. It may be applied to simultaneous detection of NEO and 8 QNS, including NOR, enoxacin, ciprofloxacin, ofloxacin, fleroxacin, marbofloxacin, enrofloxacin, and pefloxacin. Graphical abstract Schematic of a lateral flow assay (LFA) based on an indirect competitive model. By using two test lines, the LFA can detect the neomycin and quinolones antibiotics simultaneously. Based on the surface-enhanced Raman scattering (SERS), the LFA shows high sensitivity to antibiotics with low limit of detection.

Visual single cell detection of dual-pathogens based on multiplex super PCR (MS-PCR) and asymmetric tailing HCR (AT-HCR)

To prevent the propagation of pathogens and minimize the damage to humans, an ultrasensitive and colorimetric dual detection method for pathogens was developed and validated based on the Multiplex Super Polymerase Chain Reaction (MS-PCR) and Asymmetric Tailing Hybridization Chain Reaction (AT-HCR). In the 5-min MS-PCR, dual pathogens were recognized and amplified simultaneously, generating plenty of dsDNA products with protruding ssDNA toeholds. Since a blocker of oxyethyleneglycol bridge was exquisitely inserted into two-set forward primers, which hindered the polymerase extension. Then, two-set ssDNA toeholds initiated two-set AT-HCR in 20-min and 60-min visual analysis depending on G-quadruplex DNAzyme was induced by Terminal deoxynucleotidyl Transferase (TdT) at the tails of AT-HCR at 3′-OH. During the process, the first magnetic separation was conducted after MS-PCR based on our home-made XP beads, to remove primer-dimers and other impurities in 30-min. The second magnetic separation was performed before TdT catalysis based on dynabeads, to eliminate unreacted AT-HCR hairpins in 15-min. To challenge the practical application capability of this strategy, the detection of analyte in fat-free milk was also tested and demonstrated similar detection capability to visualize single cell. Therefore, the proposed sensor exhibited a high selectivity and sensitivity and is thus applicable for visual single cell detection of dual-pathogens of Salmonella spp. and Staphylococcus aureus simultaneously in 130-min. Generally, it provides a visual, ultrasensitive, rapid and universal detective method for dual-target analysis.

Optofluidic gutter oil discrimination based on a hybrid-waveguide coupler in fibre.

Discriminating edible oils from gutter oils has significance in food safety, as illegal gutter oils cannot meet a variety of criteria such as the acid value, peroxide value and quality. To discriminate these illegal cooking oils, we propose an ultrasensitive optofluidic detection method based on a hybrid-waveguide coupler. Prior to the straight waveguide inscription in the cladding of the silica tube using a femtosecond laser, a section of coreless fibre is firstly spliced with the ST to supply a platform for the inscription of an S-band waveguide. Then a pair of microfluidic channels are ablated on the ST using the fs laser to enable liquid analytes to flow in and out of the air channel. In the transmission spectrum, a unique resonant loss dip can be observed, which is produced by coupling the light from the laser inscribed waveguide to the liquid core when the phase-matching condition is met. This hybrid-waveguide coupler with a simplified structure realizes dynamic optofluidic refractive index sensing with an ultrahigh sensitivity of -112 743 nm RIU-1, a detection limit of 2.08 × 10-5 RIU and a refractive index detection range from 1.4591 to 1.4622. This novel method can be used for food safety detection, specifically, for the discrimination of gutter oils.

Ultrasensitive DNA detection based on target-triggered rolling circle amplification and fluorescent poly(thymine)-templated copper nanoparticles.

We describe a novel strategy for the ultrasensitive detection of target DNA based on rolling circle amplification (RCA) coupled with fluorescent poly(thymine)-templated copper nanoparticles (poly T-CuNPs). In the presence of target DNA, a padlock DNA probe that consists of two regions: a target DNA-specific region and a poly(adenine) region, is circularized by the ligation reaction, and the subsequent RCA reaction is promoted to generate long, concatemeric, single-stranded DNA (ssDNA) with a lot of repetitive poly T sequences. As a result, a large number of poly T-CuNPs are formed, exhibiting a highly fluorescent signal. However, in the absence of target DNA or in the presence of non-specific target DNA, the padlock DNA probe is not circularized and the subsequent RCA is not executed, leading to no production of fluorescent poly T-CuNPs. With this simple strategy, we successfully analyzed the target DNA with the ultralow detection limit of 7.79 aM, a value that is 3 or 7 orders of magnitude lower than those of previous RCA-based fluorescent DNA detection strategies. In addition, the developed system was demonstrated to selectively discriminate non-specific target DNAs with one-base mismatch, suggesting potential application in the accurate diagnosis of single nucleotide polymorphisms or mutations.

Primer dephosphorylation-initiated circular exponential amplification for ultrasensitive detection of alkaline phosphatase.

Alkaline phosphatase (ALP) is an important diagnostic indicator for various human diseases including bone diseases, liver dysfunction, diabetes, breast and prostatic cancers. However, the conventional methods for ALP assay are usually cumbersome and time-consuming with low sensitivity. Here, we develop a new fluorescent method for ultrasensitive detection of ALP activity on the basis of primer dephosphorylation-initiated isothermal circular exponential amplification. We design two dual-functional hairpin probes (HP1 and HP2), which function as both the templates for exponential amplification reaction (EXPAR) and the generators for signal output. In the presence of ALP, the 3'-phosphorylated primer is dephosphorylated and subsequently hybridizes with the 3' protruding end of HP1 to initiate the first strand displacement amplification (SDA), producing trigger 1 and fluorescence signal. The released trigger 1 is complementary to the 3' protruding end of HP2 for the initiation of the second SDA, producing trigger 2 and fluorescence signal. Notably, trigger 2 is complementary to the 3' protruding end of HP1 and may subsequently initiate two consecutive SDAs, enabling circular EXPAR to generate an amplified fluorescence signal. This method exhibits high sensitivity with a detection limit of 2.0 × 10-10 U μL-1 and a large dynamic range of 5 orders of magnitude from 1.0 × 10-9 to 1.0 × 10-4 U μL-1, and it can measure ALP at the single-cell level. Importantly, this method can be applied for the measurement of kinetic parameters and the screening of potential inhibitors, providing a powerful tool for ALP-related biomedical research and clinical diagnosis.

N-Carbamoylmaleimide-treated carbon dots: stabilizing the electrochemical intermediate and extending it for the ultrasensitive detection of organophosphate pesticides.

To date, numerous methods have been reported for the detection of organophosphorus pesticides (OP) due to their severe potential hazard to the environment, public health and national security. However, very few works have ever found that the signal loss of thiocholine (TCh) during electrochemical processing is a key factor leading to the low sensitivity of acetylcholinesterase (AChE)-based OP electrochemical sensing platforms. Herein, we propose an ultrasensitive detection method for multiple OPs including parathion-methyl, paraoxon, dimethoate and O,O-dimethyl-O-2,2-dichlorovinyl-phosphate using N-carbamoylmaleimide-functionalized carbon dots (N-MAL-CDs) as a nano-stabilizer. For the first time, Michael addition is introduced into an AChE-based OP electrochemical sensing platform to enrich the electrochemical intermediate TCh. The Michael addition between TCh and N-MAL-CDs is demonstrated via XRD, FTIR, SEM and EDS elemental mapping experiments. Due to the stabilization and enhancement of TCh with N-MAL-CDs, the as prepared OP sensing platform achieves ultrahigh sensitivity by detecting the initial electrochemical signals of TCh without signal loss, showing a wide linear range of 3.8 × 10-15-3.8 × 10-10 M for parathion-methyl and 1.8 × 10-14-3.6 × 10-10 M for paraoxon, with a limit of detection of 1.4 × 10-15 M for parathion-methyl and 4.8 × 10-15 M for paraoxon.

Development of an Ultra-sensitive Detection Method for Genetically Modified Soybeans in Natto, a Traditional Japanese Fermented Food

Development of an Ultra-sensitive Detection Method for Genetically Modified Soybeans in Natto, a Traditional Japanese Fermented Food

Copper (II) oxide nanozyme based electrochemical cytosensor for high sensitive detection of circulating tumor cells in breast cancer

An ultrasensitive electrochemical detection method was developed to detect circulating tumor cells (CTCs) by using reduced graphene oxide/gold nanoparticles composites (rGO/AuNPs composites) as a support material with CuO nanozyme as a catalyst. MCF-7 circulating tumor cells were detected by an electrochemical cytosensor with effective surface recognition between specific mucin 1 protein (MUC-1) over-expressed on the MCF-7 cell membranes and MUC-1 aptamer. The CuO nanozyme is used as a signal-amplifying nanoprobe in an ultrasensitive electrochemical cytosensor to detect CTCs for the first time. Under the optimized experimental conditions, the proposed cytosensor exhibited significant analytical performance for the determination of MCF-7 circulating tumor cells. A wide detection range from 50 to 7×103cellsmL−1 with a detection limit as low as 27cellsmL−1 was reached on the condition of acceptable selectivity and reproducibility. Furthermore, the cytosensor can easily distinguish CTCs from the real serum sample due to the specific combination of MUC-1 and MUC-1 aptamer.