Report Probes Research Articles

A universal nucleic acid detection platform combing CRISPR/Cas12a and strand displacement amplification with multiple signal readout

Rapid and sensitive detection of nucleic acids has become crucial in various fields. However, most current nucleic acid detection methods can only be used in specific scenarios, such as RT-qPCR, which relies on fluorometer for signal readout, limiting its application at home or in the field due to its high price. In this paper, a universal nucleic acid detection platform combing CRISPR/Cas12a and strand displacement amplification (CRISPR-SDA) with multiple signal readout was established to adapt to different application scenarios. Nucleocapsid protein gene of SARS-CoV-2 (N gene) and hepatitis B virus (HBV) DNA were selected as model targets. The proposed strategy achieved the sensitivity of 53.1 fM, 0.15 pM, and 1 pM for N gene in fluorescence mode, personal glucose meter (PGM) mode and lateral flow assay (LFA) mode, respectively. It possessed the ability to differentiate single-base mismatch and the presence of salmon sperm DNA with a mass up to 105-fold of the targets did not significantly interfere with the assay signal. The general and modular design idea made CRISPR-SDA as simple as building blocks to construct nucleic acid sensing methods to meet different requirements by simply changing the SDA template and selecting suitable signal report probes, which was expected to find a breadth of applications in nucleic acids detection.

Signal-on low background electrochemiluminescence biosensor based on the target-triggered dual signal amplifications and difference of electrostatic attraction

The electrostatic attraction difference between negatively charged report probes and positively charged electrode had been applied to develop a signal-on low background electrochemiluminescence (ECL) biosensor for microRNA-141 (miRNA141, chosen as a model target) detection. Indium tin oxide (ITO) electrode was modified with poly-(allylamine hydrochloride) (PAH) to generate a positively charged surface. The electrostatic repulsion between dichlorotris (1,10-phenanthroline) ruthenium (Ⅱ) hydrate (Ru(phen)32+) and ITO electrode results in the low background signal. The target can trigger an enzyme-free isothermal catalytic hairpin self-assembly (CHA) and hybrid chain reaction (HCR) cascade reaction to generate a large amount of negatively charged long dsDNAs, which can be used to load Ru(phen)32+ (Ru-dsDNA) with high efficiency. Ru-dsDNA complexes are negatively charged and can easily accumulate on ITO electrode surface because of electrostatic attraction, which results in a strong ECL signal detected. The ECL intensity of the system was linearly correlated with the logarithm of microRNA-141 concentration in the range of 1 fM ∼ 5 pM with a detection limit of 0.17 fM (S/N = 3). The reliability of the proposed biosensor had been verified through the detection of targets in cell lysates.

In situ fabrication of urchin-like Cu@carbon nanoneedles based aptasensor for ultrasensitive recognition of trace mercury ion

Mercury ion (Hg2+) is a strong toxic heavy ion that causes severe damages to the environment and readily accumulates in the food chain. However, it remains a major challenge to realize a sensitive and precise recognition of Hg2+ with a trace concentration for early identifying the pollution source. In this work, a novel electrochemical aptasensor was designed to achieve an ultrasensitive and quantitative detection of trace Hg2+, relying on an urchin-like architecture of Cu@carbon nanoneedles (Cu@CNNs) as the electroactive probe. This specific nanostructure was in-situ constructed through a controllable pyrolysis process, serving as a signal magnifier and DNA loading platform owing to its outstanding electrocatalysis and large specific surface areas. Meanwhile, an exonuclease III-assisted cycling amplification strategy was designed to efficiently amplify the signal strength of trace Hg2+via the consecutive release of report probes in nicking reaction. This as-prepared Hg2+ aptasensor exhibited an ultralow detection limit of 3.7 fM (7 × 10−6 ppm) and a wide linear range from 10 fM to 10 μM, together with the satisfactory stability and reusability for assay in real water samples. It is highly expected that this Cu@CNNs based aptasensor will have tremendous potentials in the early warning and efficient pollution monitoring of heavy metal ions.

An encodable multiplex microsphere-phase amplification sensing platform detects SARS-CoV-2 mutations

SARS-CoV-2 variants of concern (VOCs) contain several single-nucleotide variants (SNVs) at key sites in the receptor-binding region (RBD) that enhance infectivity and transmission, as well as cause immune escape, resulting in an aggravation of the coronavirus disease 2019 (COVID-19) pandemic. Emerging VOCs have sparked the need for a diagnostic method capable of simultaneously monitoring these SNVs. To date, no highly sensitive, efficient clinical tool exists to monitor SNVs simultaneously. Here, an encodable multiplex microsphere-phase amplification (MMPA) sensing platform that combines primer-coded microsphere technology with dual fluorescence decoding strategy to detect SARS-CoV-2 RNA and simultaneously identify 10 key SNVs in the RBD. MMPA limits the amplification refractory mutation system PCR (ARMS-PCR) reaction for specific target sequence to the surface of a microsphere with specific fluorescence coding. This effectively solves the problem of non-specific amplification among primers and probes in multiplex PCR. For signal detection, specific fluorescence codes inside microspheres are used to determine the corresponding relationship between the microspheres and the SNV sites, while the report probes hybridized with PCR products are used to detect the microsphere amplification intensity. The MMPA platform offers a lower SARS-CoV-2 RNA detection limit of 28 copies/reaction, the ability to detect a respiratory pathogen panel without cross-reactivity, and a SNV analysis accuracy level comparable to that of sequencing. Moreover, this super-multiple parallel SNVs detection method enables a timely updating of the panel of detected SNVs that accompanies changing VOCs, and presents a clinical availability that traditional sequencing methods do not.

Old commercialized magnetic particles new trick: Intrinsic internal standard

Magnetic particles (MPs) are the most widely used commercialized engineering particles, which gained great success in various biological applications. Inspired by their intrinsic Fe isotope composition, we discovered a commercialized MPs-internal standard's novel function to realize the accurate quantification of biomolecules. The bioassay of carcinoembryonic antigen (CEA) was chosen as a modal system. The Fe isotope in MPs and Au isotope in report probes were simultaneously and sensitively detected by the elemental mass spectrometry. 197Au/57Fe isotopic ratios and CEA concentrations showed good linearity in the range of 0.6–300 ng/mL, with a detection limit of 0.09 ng/mL (3σ). The accuracy and precision of the proposed MPs-based immunoassay were greatly improved, by eliminating potential MPs loss during magnetic separation and absolute intensity fluctuations. Considering the exceptional availability and universality of commercialized MPs, the proposed method might open a new avenue for MPs' biological applications.

An ultrasensitive reusable aptasensor for noninvasive diabetic retinopathy diagnosis target on tear biomarker

A sensitive, non-invasive and specific tear biomarker sensor for diabetic retinopathy (DR) could be of great significance in clinical practice. Vascular endothelial growth factor (VEGF) was targeted as a tear biomarker for DR. In principle, we used hybridization chain reaction (HCR) and CeO2 nanoparticles as a cascade signal amplification strategy to construct a reusable aptasensor in search of VEGF. The report probes H1 and H2 modified with medium CeO2 nanoparticles were added in and HCR reaction. The first analysis method for a quantitative detection of VEGF was obtained using CeO2 nanoparticles to catalyze H2O2 to generate electrical signals. The fabricated biosensor exhibited an ultrasensitive detection of VEGF down to 0.27 fg mL−1, response range from 1 fg mL−1 to 0.1 ng mL−1. Subsequently the aptasensor carried out a second repeated assembly to obtain a second signal by strand displacement reaction (SDR). The ratio of the former and latter signals was used to carry out a second analysis method for quantifying VEGF. The second analysis method had detection limits as low as 7.39 fg mL-1, ranging from 10 fg mL−1 to 0.1 ng mL−1. The aptasensor could maintain above 99 % signal in five days and shows the acceptable reproducibility. For a practical tear biomarker detection, the analysis of the tears was carried out simultaneously using ELISA and the aptasensor which showed excellent precision. This non-invasive screening method provides a potential tool for the early diagnosis and monitoring of DR and may improve screening success among high-risk patients with diabetes.

Portable detection of colorectal cancer SW620 cells by using a personal glucose meter

It is of great value to develop general, low-cost and even household methods for colorectal cancer detection. Here, a portable detection strategy based on a personal glucose meter (PGM) was designed for meeting this purpose. In this strategy, the anti-EpCAM coated magnet beads (MBs) were used as capture probes for enriching cancer cells and the aptamer modified and invertase loaded graphene oxides (GO) were used as report probes for producing glucose signal. This method is sensitive with detection limit as low as 560 cells, and demonstrates excellent detection specificity. Meanwhile, we succeeded in the specific detection of target cells in 20% human serum samples, indicating this method has great prospect in clinical diagnosis. Moreover, this method presents favourable universality for detecting different colorectal cancer cells by just using different recognition aptamers. Importantly, this method can be implemented for the target cell detection at room temperature without any expensive and large-scale instruments but a portable PGM. Therefore, this portable detection method possesses great potential in point-of-care detection of colorectal cancer cells.

High-density micro-well array with aptamer-silver conjugates for cell sorting and imaging at single cells

Characterizing cell behavior is important to modern medical diagnoses as the changes of cell behavior are often indicators of huge diseases. In order to gain enough information about cells, developing novel methods of cell sorting and imaging is an important task. With development of micro-fabrication technologies, more advanced miniaturized devices are applied to cell research. Here, a portable and easy-to-use chip with high-density periodic micro-well array is designed and fabricated to capture target cells specifically. Combining with aptamer-silver conjugates and FAM functioned report probes, the sandwich assay was successfully applied for imaging cells. Any well of the chip is carefully designed to provide abundant information on single cells. Since there are 19,200 microwells in a single chip, more information is available. Compared to other cells, such as HEK-293, MCF-7, U2OS and Ramos cells, the sandwich assay shows high specificity towards target cell CCRF-CEM. What's more, the applications of the chip can be further expanded to other cells imaging if suitable aptamers were selected. This high-density micro-well array of aptamer-silver conjugates is hopeful to play an important role in medical diagnosis in the future.

Isothermal Microcalorimetry: Insight into the Impact of Crystallite Size and Agglomeration on the Lithiation of Magnetite, Fe3O4.

Magnetite, Fe3O4, holds significant interest as a Li-ion anode material because of its high theoretical capacity (926 mAh/g) associated with multiple electron transfers per cation center. Notably, both crystallite size and agglomeration influence ion transport. This report probes the effects of crystallite size (12 and 29 nm) and agglomeration on the reactions involved with the formation of the surface electrolyte interphase on Fe3O4. Isothermal microcalorimetry (IMC) was used to determine the parasitic heat evolved during lithiation by considering the total heat measured, cell polarization, and entropic contributions. Interestingly, the 29 nm Fe3O4-based electrodes produced more parasitic heat than the 12 nm samples (1346 vs 1155 J/g). This observation was explored using scanning electron microscopy (SEM) and X-ray fluorescence (XRF) mapping in conjunction with spatially resolved X-ray absorption spectroscopy (XAS). SEM imaging of the electrodes revealed more agglomerates for the 12 nm material, affirmed by XRF maps. Further, XAS results suggest that Li+ transport is more restricted for the smaller crystallite size (12 nm) material, attributed to its greater degree of agglomeration. These results rationalize the IMC data, where agglomerates of the 12 nm material limit solid electrolyte interphase formation and parasitic heat generation during lithiation of Fe3O4.

Ratiometric quantification of β2-microglobulin antigen in human serum based on elemental labeling strategy

Ratiometric quantification for competitive immunoassay based on internal standard element detection utilizing inductively coupled plasma mass spectrometry (ICP-MS) as multiplex readout has been demonstrated. The Beta-2-microglobulin (β2-MG) associated with clinical diseases was detected by Y-labeled capture antibody used as internal standard probes and Sm-labeled antigen used as report probes via antigen-antibody reaction. The ratiometric quantification was achieved by taking the signal ratio of Sm/Y. The ratiometric method could compensate for the particle loss and suppress the signal fluctuation, which improved the precision of the quantitative result. Under the optimized conditions, the calibration curves for β2-MG antigen was linear in the range of 0.25–8.0 μg/mL with a detection limit of 0.17 μg/mL (3σ, n = 11). The recoveries of 96.5%–132% were obtained for serum samples spiked with different concentration standards, and the relative standard deviation (RSD) was less than 10%. The β2-MG results in serum samples obtained by the proposed method correlated well with those obtained by time-resolved fluorescence immunoassay (r = 0.947). This proposed method provides a simpler labeling strategy for ratiometric quantification of immunoassay.

Fluorescent and colorimetric dual-mode aptasensor for thrombin detection based on target-induced conjunction of split aptamer fragments

Since the lack of detection diversity of the single-signal readout strategy, it is urgent to develop fast and multisignal assay strategies. A highly selective and sensitive assay method with colorimetric and fluorometric dual signals readouts is presented in this paper. It is based on the principle that the target induced conjunction of split aptamer fragments assembled on the surface of Au nanoparticles (AuNPs). In the presence of targets, the color of solution changed from wine red to blue and can be measured both visual inspection and spectrophotometry because of the aggregation of AuNPs. At the same time, the report probes which are original hybrid with the anchoring aptamer fragments on the AuNPs surface can be released and recovers the fluorescence. By use of this detection strategy, the limit of detection for thrombin (TMB), as a model of analyte, were 0.45 and 0.16nM, respectively. Furthermore, this protocol can discriminate TMB from other analogue with high selectivity and can be used to detect TMB in human serum samples. The results came from the two signals were well consistent with each other, which demonstrated that it has application potential for detection of TMB in complex matrix.

A highly sensitive and reusable electrochemical mercury biosensor based on tunable vertical single-walled carbon nanotubes and a target recycling strategy.

In this work, a novel electrochemical Hg2+ biosensor with high sensitivity and excellent reusability was presented. The sensor was based on tunable vertical single-walled carbon nanotubes (v-SWCNTs) and a target recycling strategy. A facile and scalable approach involving the conformational regulation of self-assembled monolayers was established for the fabrication of v-SWCNTs with tailored orientation and homogeneity. The obtained v-SWCNTs exhibited superior properties including a large specific area, high electrical conductivity, and excellent substrate binding strength, opening up a wide horizon for advanced electrochemical applications. Meanwhile, an efficient Hg2+ recycling strategy was designed using exonuclease III. In this strategy, a trace amount of Hg2+ triggered consecutive nicking reactions, and numerous report probes were released to bind with v-SWCNTs through π-π interactions. Based on the innovative design, an ultralow detection limit of 3 fM (S/N = 3), a wide linear range from 10 fM to 1 μM, high selectivity, and good reliability were achieved for a Hg2+ assay in water and serum samples using the prepared biosensor. Besides, due to the reversibility of π-π interactions, the stable v-SWCNT interface was regenerated for 50 consecutive measurements without obvious signal loss, making it a promising candidate for routine and efficient Hg2+ monitoring.

A facile “turn-on” fluorescent method with high sensitivity for Hg2+ detection using magnetic Fe3O4 nanoparticles and hybridization chain reactions

In this manuscript, the authors molecularly engineered a hybridization chain reactions (HCRs) based probe on magnetic Fe3O4 nanoparticles for the sensitive detection of Hg2+. The sensing system comprised three probes: capture probe H1, report probe H2, and report probe H3. The capture probe was modified on the surface of magnetic Fe3O4 nanoparticles. The report probes were labeled with fluorescein isothiocyanate (FITC). Without Hg2+, the report probes were stable as molecular beacons in solution. In the presence of Hg2+, the T-rich capture probes and report probes will hybridize into double-helical DNA domains with the aid of T–Hg2+–T coordination chemistry. Trigged by this reaction, more molecular beacons open and form a super tandem structure. Herein, the fluorescence signal was magnified by capturing more report probes. Separating the target and captured report probes from reaction solution was benefit to decrease the background signal and interference from other metal ions. The detection limit of this method was about 0.36nM, which is much lower than the regulations of World Health Organization and U.S. Environmental Protection Agency on Hg2+ in drink water. This proposed sensing strategy also showed favorable selectivity over other common metal ions. In addition, it has good practicability in real water samples.

Surface plasmon resonance biosensor for enzyme-free amplified microRNA detection based on gold nanoparticles and DNA supersandwich

A novel enzyme-free amplified surface plasmon resonance (SPR) biosensor for microRNA (miRNA) detection was developed based on gold nanoparticles (AuNPs) coupled with DNA supersandwich. In the detection strategy, the DNA-linked AuNPs as the primary amplification element, not only hybridized with the capture DNA on the Au film to amplify SPR signal but also initiated the subsequent secondary amplification, i.e. DNA supersandwich formation of two report probes. In the presence of target, stem–loop structure of capture DNA on the Au film surface was unfolded, and DNA-linked AuNPs were bound to Au film by hybridization with terminus of capture DNA. Then, the carried assistant DNA on the AuNPs could trigger an alternative hybridization reaction of two report probes, resulting in the formation of DNA supersandwich. Due to the electronic coupling between localized plasmon of AuNPs and the surface plasmon wave associated with Au film, as well as the enhancement of the refractive index of the medium next to the metal film caused by DNA supersandwich structure, the shift of resonance angle was enhanced obviously. By employing the enzyme-free dual signal amplification strategies, as low as ca. 8fM miRNA-21 could be detected. Moreover, this assay also showed high selectivity toward single-base mismatch, and demonstrated its applicability for the target detection in human serum. This work may provide great potential applications in future clinical analysis.

A novel ultrasensitive electrochemical DNA sensor based on double tetrahedral nanostructures.

Electrochemical DNA (E-DNA) sensor is an important tool for detecting DNA biomarker. In this work, we have demonstrated a novel strategy of E-DNA sensor based on DNA tetrahedral nanostructures for the sensitive detection of target DNA. In our design, thiol and biotin modified DNA tetrahedral nanostructures were used as capture and report probes respectively. The biotin-tagged three dimensional DNA tetrahedral nanostructures were employed for efficient signal amplification by capturing multiple catalytic enzymes. Such improved E-DNA sensor can sensitively detect DNA target as low as 1 fM with excellent differentiation ability for even single mismatch. And a mean recovery rate of 90.57% in DNA solution extracted from human serum was obtained. We have also compared this new method of attaching catalytic enzymes with the other two typical methods: One is through biotinylated single-stranded DNA (SSDNA) and the other is through gold nanoparticles (GNPs). Results indicated that the RTSPs-based enzyme amplification system showed much better performance than the other two systems.

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A novel sandwich assay with molecular beacon as report probe for nucleic acids detection on one-dimensional microfluidic beads array

A novel sandwich assay with molecular beacons as report probes has been developed and integrated into one-dimensional microfluidic beads array (1-D chip) to pursue a label-free and elution-free detection of DNA/mRNA targets. In contrast with the immobilized molecular beacons, this sandwich assay can offer lower fluorescence background and correspondingly higher sensitivity. Furthermore, this sandwich assay on 1-D chip operating in conjunction with molecular beacon technique allows multiple targets detection without the need of laborious and time-consuming elution, which makes the experiment process simple, easy to handle, and reproducible results. In the experiment, the synthesized DNA targets with different concentrations were detected with a detection limit of ∼0.05 nM. Moreover, the mRNA expression changes in A549 cells before and after anticancer drug 5-flouorouracil treatments were detected and the results were validated by the conventional RT-PCR method.

South African Experience of Gender and Affirmative Action: A Research Report

This research report is based on a study carried out to examine the degree of effectiveness of affirmative action in reversing the ills suffered by women in general and Black women in particular on account of apartheid. The main objective is to probe the operationalization of affirmative action in the context of South Africa and to consider if its underlying logic directed specifically at Black women is sufficiently strong to deal with the issue of gender inequality which intersects with other race and class dynamics. Rather than focusing primarily on the experience of Black women the research report probes into the ways affirmative action has been formulated planned initiated implemented and co-ordinated by various agencies and departments of government. The report is based on the view that the ultimate judgment as to the effectiveness and success or otherwise of affirmative action to respond to the challenges of the past and to meet the needs of the previously underprivileged and its ability to foster and innovate ideas that support appropriate intervention strategies remains with the government itself. In 1995 the government of South Africa conducted a socio-economic survey to identify the ways in which the measures adopted in relation to affirmative action have contributed to the understanding of the lives of Black South African women. (excerpt)

The regulation of β-secretase by cholesterol and statins in Alzheimer's disease

Epidemiologists have found a decreased risk of developing Alzheimer's disease (AD) in people taking statins (cholesterol biosynthesis inhibitors). We have reported previously that, in cell culture, lovastatin decreases the output of β-amyloid, a peptide that is toxic to neurones, and is reputably the prime cause of neurodegeneration seen in AD. This report probes the mechanism of statin protection further by finding out how the protease β-secretase, that releases β-amyloid from its precursor protein, behaves under changed cholesterol levels induced by statins. We found that, with high cellular cholesterol levels, there is a decrease in glycosylation of mature oligosaccharides in β-secretase, whereas in the presence of lovastatin, glycosylation progresses further. Moreover, lovastatin does not inhibit β-secretase in vitro. Thus, the cholesterol and statin effects are due to changes in cellular targeting induced by changed cholesterol gradients. Some of these changes are mimicked by the action of U18666A, a cholesterol-transport inhibitor that produces a defect in cells seen in patients with Neimann Pick's disorder.

Quantification of Splice Variants Using Molecular Beacon or Scorpion Primers

Uncovering the relationship between the generation of alternative transcripts and cellular processes is of crucial importance in the exploration of a gene's biology. The description and quantification of the spatiotemporal splicing pattern can be one method to select the most interesting transcripts for future studies. Fluorescence-based real-time quantitative RT-PCR has recently revolutionized the possibilities for transcriptional quantification studies. In this report, Molecular Beacon and Scorpion probes have been tested as new possibilities for determining the expression level of alternative transcripts. We validated these systems by analyzing alternative splicing of exons 6, 15, and 16 of the calpain 3 gene with tissues containing large variation in the ratio of the different transcripts. We determined conditions that demonstrated that boundary probes are useful tools and good alternatives to boundary primers, when developing a system to quantify specific transcripts. We suggest that the choice of a quantification system should depend in part on the structure and base composition of the gene and may have to be determined experimentally.