Ultrasensitive Identification Research Articles

The trace detection of Hg2+ by flexible electrochemical biosensor based on covalent organic framework synergistic amplified strategy

Mercury (II) ion (Hg2+) is highly toxic, which is harmful to the environment and human health. Hg2+ electrochemical detection method lacks high sensitivity and convenience. Thus, we fabricated a new-style flexible sandwich biosensor for ultra-sensitive identification of trace mercury ions in water. A large number of gold nanoparticles (AuNPs) were electrodeposited on the flexible electrode as the sensing interface, and the Au-functionalized covalent organic framework (COF) nanocomposites adsorbed a large amount of methylene blue (MB) were used as the signal amplifier. COF/Au provided a large specific surface area, which was easier to assemble with mercaptan functionalized capture DNAs (cDNA) aptamers via S-Au bonds for selective recognition of mercury, to improve the conductivity and electrochemical performance. Meanwhile, gold nanoparticles (AuNPs), MB and reporter DNA (rDNA) were anchored to COF to fabricate the COF/Au/MB/rDNA probe. The prepared COF based probe exhibited a large surface area, excellent electrical signal response, and good environmental compatibility. After incubation with Hg2+, rDNA was tightly bound to cDNA via thymidine-Hg2+-thymine pairing (T-Hg2+-T). Thus, the COF based probe was specifically connected to the flexible interface to generate electrical signals. Experimental results indicated that the biosensor exhibited wider linear response range of 0.1 pM to 100 nM Hg2+, and lower detection limit of 45.2 fM (3σ/k). In addition, the prepared ligand sensor also showed reliable detection performance in real water samples.

Uncovering Art's Vanishing Hues with Surface-Enhanced Raman Scattering: Drawing Inspiration from the Past for the Future.

The aesthetic and historical significance of art is well recognized; art can stoke emotions, invite close inquiry, and connect us to the past. However, works of art are also complex material objects that present unique challenges and opportunities for the scientific community. Identifying "fugitive" organic pigments in traditional oil paintings, for example, presents a particularly complex analytical challenge that is critical to address for their conservation and long-term preservation. In this Perspective, we discuss the benefits and technical challenges of applying surface-enhanced Raman scattering (SERS) spectroscopy to the ultrasensitive identification of fugitive pigments in paintings as well as future developments in SERS we envision that are inspired by the past.

DNA-Templated Click Ligation Chain Reaction Catalyzed by Heterogeneous Cu2O for Enzyme-Free Amplification and Ultrasensitive Detection of Nucleic Acids.

Nucleic acids play a pivotal role in the diagnosis of diseases. However, rapid, cost-efficient, and ultrasensitive identification of nucleic acid targets still represents a significant challenge. Herein, we describe an enzyme-free DNA amplification method capable of achieving accurate and ultrasensitive nucleic acid detection via DNA-templated click ligation chain reaction (DT-CLCR) catalyzed by a heterogeneous nanocatalyst made of Cu2O (hnCu2O). This hnCu2O-DT-CLCR method is built on two cross-amplifying hnCu2O-catalyzed DNA-templated azide-alkyne cycloaddition-driven DNA ligation reactions that boast a fast reaction rate and a high DNA ligation yield in minutes, enabling rapid exponential amplification of specific DNA targets. This newly developed hnCu2O-DT-CLCR-enabled DNA amplification strategy is further integrated with two signal reporting mechanisms to achieve low-cost and easy-to-use biosensors: an electrochemical sensor through the conjugation of a methylene blue redox reporter to a DNA probe used in hnCu2O-DT-CLCR and a colorimetric sensor through the incorporation of the split-to-intact G-quadruplex DNAzyme encoded into hnCu2O-DT-CLCR. Both sensors are able to achieve specific detection of the intended DNA target with a limit of detection at aM ranges, even when challenged in complex biological matrices. The combined hnCu2O-DT-CLCR and sensing strategies offer attractive universal platforms for enzyme-free and yet efficient detection of specific nucleic acid targets.

Clinical utility of an ultra-sensitive ctDNA NGS assay for the detection and monitoring in HR-positive, HER2-negative breast cancer.

e13076 Background: Breast cancer is the most diagnosed cancer in women globally, mainly as hormone receptor HR-positive, HER2-negative. While endocrine therapies have markedly improved clinical outcomes, the threat of disease progression persists. This study introduces an ultra-sensitive circulating tumor DNA (ctDNA) Next-Generation Sequencing (NGS) assay that was designed for in-depth genomic profiling of breast cancer patients undergoing endocrine therapy, aimed at detecting treatment resistance and monitoring disease progression. Methods: In this ongoing investigation, 46 patients with advanced HR-positive, HER2-negative breast cancer were enrolled, and plasma samples were collected subsequent to first-line aromatase inhibitor (AI) treatment and before the initiation of second-line treatment with fulvestrant. The study employed the PredicineCARE ULTRA, an ultra-sensitive liquid biopsy assay with a proprietary NGS panel and exceeds 100,000x coverage, allowing for the ultra-sensitive identification of genomic alterations, surpassing the detection capabilities of standard ctDNA NGS assays with approximately 20,000x coverage. Results: Within the cohort of 46 patients, the ultra-sensitive assay detected 193 somatic mutations, 95 gene copy number variants (CNVs), and a gene fusion involving FGFR3. The most commonly mutated genes, with a prevalence of 20% or more, were TP53 (33%), PIK3CA (26%), ATM (24%), ESR1 (22%), and BRCA2 (20%). These findings were benchmarked against results from a down-sampling algorithm designed to emulate ctDNA assays with 20,000x coverage. The ultra-sensitive assay revealed 31.3% more mutations (193 vs. 147) and 30.1% more CNVs. It was especially proficient in detecting mutations with allele frequencies (MAF) below 0.1%, identifying 10 additional mutations not observed at the standard ctDNA assay sensitivity, with the lowest MAF detected being 0.03% compared to the 0.1% threshold of the standard assay. This heightened sensitivity was evident in genes associated with targeted therapies and drug resistance, identifying 30.7% more variations in PIK3CA and 11.1% more in ESR1, with the lowest MAFs of 0.08% and 0.06%, respectively. The FGFR3-BAIAP2L1 gene fusion was also uniquely detected by the ultra-sensitive assay, with a MAF of 0.09%. Conclusions: The ultra-sensitive ctDNA NGS assay demonstrated superior detection of low-frequency mutations in HR-positive, HER2-negative breast cancer, significantly outperforming standard liquid biopsy assays. This enhanced sensitivity may offer profound implications for identifying more diagnostically positive patients who may benefit from targeted therapy such as PIK3CA and ESR1 inhibitors. This ultra-sensitive ctDNA assay will also enable early detection of treatment resistance and refined disease monitoring, potentially guiding more effective personalized therapies.

Quinoline Bridging Hyperconjugated Covalent Organic Framework as Solid-Phase Microextraction Coating for Ultrasensitive Determination of Phthalate Esters in Water Samples.

Phthalate esters (PAEs) are widely distributed in the environment, and this has caused serious health and safety concerns. Development of rapid and ultrasensitive identification and analysis methods for phthalate esters is urgent and highly desirable. In this work, a novel nitrogen-rich covalent organic framework (N-TTI) derived quinoline bridging covalent organic framework (N-QTTI) was fabricated and used as a solid-phase microextraction (SPME) coating for the ultrasensitive determination of phthalate esters in water samples. The physical and chemical properties of N-QTTI were investigated sufficiently. The N-QTTI-coated fiber demonstrates a superior enrichment performance than either the N-TTI-coated fiber or commercial fibers under the optimized SPME conditions. For the first time, we propose a semi-immersion strategy for the extraction of PAEs from water samples based on N-QTTI-coated SPME fibers. Combined with gas chromatography-mass spectrometry (GC-MS), the developed method N-QTTI-SPME-GC-MS exhibits a wide linear range with a satisfactory linearity (R2 ≥ 0.995). The limits of detection (LOD, S/N = 3) and the limits of quantification (LOQs, S/N = 10) were 0.17-1.70 and 0.57-5.60 ng L-1, respectively. The repeatability of the new method was examined using relative standard deviations (RSDs) between intraday and interday data, which were 0.38-7.98% and 1.22-6.60%, respectively. The spiked recoveries at three levels of 10, 100, and 1000 ng L-1 were in the range of 90.0-106.2% with RSDs of less than 7.48%. The enrichment factors ranged from 291 to 17180. When compared to previously published works, the LODs of the newly established method were improved 5-5400 times, and the enrichment factors were increased by at least 8 times. The absorption mechanism was investigated by X-ray photoelectron spectroscopy and noncovalent interaction force analysis. The technique was successfully employed for detecting PAEs in water samples.

Multi-Body Biomarker Entrapment System: An All-Encompassing Tool for Ultrasensitive Disease Diagnosis and Epidemic Screening.

Ultrasensitive identification of biomarkers in biofluids is essential for the precise diagnosis of diseases. For the gold standard approaches, polymerase chain reaction and enzyme-linked immunosorbent assay, cumbersome operational steps hinder their point-of-care applications. Here, a bionic biomarker entrapment system (BioES) is implemented, which employs a multi-body Y-shaped tetrahedral DNA probe immobilized on carbon nanotube transistors. Clinical identification of endometriosis is successfully realized by detecting an estrogen receptor, ERβ, from the lesion tissue of endometriosis patients and establishing a standard diagnosis procedure. The multi-body Y-shaped BioES achieves a theoretical limit of detection (LoD) of 6.74 aM and a limit of quantificationof 141aM in a complex protein milieu. Furthermore, the BioES is optimized into a multi-site recognition module for enhanced binding efficiency, realizing the first identification of monkeypox virus antigen A35R and unamplified detection of circulating tumor DNA of breast cancer in serum. The rigid and compact probe framework with synergy effect enables the BioES to target A35R and DNA with a LoD down to 991 and 0.21aM, respectively. Owing to its versatility for proteins and nucleic acids as well as ease of manipulation and ultra-sensitivity, the BioES can be leveraged as an all-encompassing tool for population-wide screening of epidemics and clinical disease diagnosis.

Photoelectrochemical biosensing platform for the SARS‐CoV‐2 spike and nucleocapsid proteins

AbstractThe COVID‐19 pandemic is still a continuing worldwide challenge for public health systems. Early and ultrasensitive identification of the infection is essential for preventing the spread of COVID‐19 by pre‐symptomatic or asymptomatic individuals, particularly in the community and in‐home settings. This work presents a versatile photoelectrochemical (PEC) immunosensor for SARS‐CoV‐2 detection based on a composite material formed by bismuth vanadate (BiVO4) and strontium titanate (SrTiO3). The PEC platform was denoted as BiVO4/SrTiO3/FTO, and it can be tuned for the detection of either Spike (S) or Nucleocapsid (N) protein by simply altering the antibody immobilized on the platform's surface. Chemical, morphological, and electrochemical characterizations were performed by X‐Ray Diffraction, Scanning Electron microscopy, Energy‐dispersive X‐ray spectroscopy, Electrochemical Impedance Spectroscopy, and Amperometry. With a simple sensing architecture of the PEC platform, it was possible to achieve a linear response range of 0.1 pg mL−1 to 1000 ng mL−1 for S protein and 0.01 pg mL−1 to 1000 ng mL−1 for N protein. The PEC immunosensors presented recovery values for the two SARS‐CoV‐2 proteins in artificial saliva samples between 97 % and 107.20 % suggesting a good accuracy for the proposed immunosensors.

One-pot, ultrasensitive, and multiplex detection of SARS-CoV-2 genes utilizing self-priming hairpin-mediated isothermal amplification

The global pandemic resulting from the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its emerging variants highlights the need for convenient and accurate detection protocols to facilitate timely prevention and management of the disease. Herein, we propose a new self-priming hairpin-mediated isothermal amplification (SIAM) protocol enabling one-pot and ultrasensitive identification of SARS-CoV-2 in a multiplexed way. This approach works by targeting a specific RNA sequence with a self-priming hairpin (SP) probe and promoting continuously repeated extension and nicking reactions to produce numerous trigger molecules, which could specifically bind to molecular beacons (MBs) and produce fluorescent signals. Under an isothermal condition of 37 °C, this technique allowed for the simultaneous identification of the spike (S) and nucleocapsid (N) genes of SARS-CoV-2 down to single copy/μL levels. We further validated the practical diagnostic capabilities of the SIAM method by accurately testing 20 clinical samples with 100% sensitivity and specificity. The SIAM method has a lot of potential to be a reliable nucleic acid testing protocol to identify infections caused by a wide range of pathogens.

Non-Hermitian Topolectrical Circuit Sensor with High Sensitivity.

Electronic sensors play important roles in various applications, such as industry and environmental monitoring, biomedical sample ingredient analysis, wireless networks and so on. However, the sensitivity and robustness of current schemes are often limited by the low quality-factors of resonators and fabrication disorders. Hence, exploring new mechanisms of the electronic sensor with a high-level sensitivity and a strong robustness is of great significance. Here, a new way to design electronic sensors with superior performances based on exotic properties of non-Hermitian topological physics is proposed. Owing to the extreme boundary-sensitivity of non-Hermitian topological zero modes, the frequency shift induced by boundary perturbations can show an exponential growth trend with respect to the size of non-Hermitian topolectrical circuit sensors. Moreover, such an exponential growth sensitivity is also robust against disorders of circuit elements. Using designed non-Hermitian topolectrical circuit sensors, the ultrasensitive identification of the distance, rotation angle, and liquid level is further experimentally verified with the designed capacitive devices. The proposed non-Hermitian topolectrical circuit sensors can possess a wide range of applications in ultrasensitive environmental monitoring and show an exciting prospect for next-generation sensing technologies.

Ultrasensitive detection and distinction of pollutants based on SERS assisted by machine learning algorithms

SERS as a promising sensing technique still faces challenges in the precise identification of trace-amount molecules due to the limitation of sensitivity and cleanliness of SERS substrate. Here, we report a precise and ultrasensitive identification of multiple pollutants via 3D clean cascade-enhanced nanosensor assisted by machine learning algorithms. This SERS substrate could achieve cascading electromagnetic energy with a remarkable enhancement factor as high as 8.35 × 109, which is attributed to the combination of micro-level polystyrene sphere (PS) porous array and nano-level Au-Ag clusters of this substrate. Benefitting from high cleanliness and ultra-sensitivity, multiple hazardous pollutants with similar geometry and Raman peaks at ultra-low concentration were successfully distinguished assisted by principal component analysis (PCA). As a result, this efficient and clean SERS substrate together with artificial intelligence could promote the application of SERS technology in the accurate identification of trace contaminants. Data AvailabilityData will be made available on request.

Programmable T-Junction Structure-Assisted CRISPR/Cas12a Electrochemiluminescence Biosensor for Detection of Sa-16S rDNA

Herein, a strand displacement amplification (SDA)-assisted CRISPR/Cas12a (LbCpf1) electrochemiluminescence (ECL) biosensor was fabricated for ultrasensitive identification of Staphylococcus aureus (Sa)-16S rDNA. A porphyrinic Zr metal-organic framework (MOF) (PCN-224) nanomaterial was prepared as the coreactant accelerator, which promoted the conversion of S2O82- and SO4*-, thus enhancing the reaction with CdS quantum dots (QDs) and amplifying the ECL emission signal. Meanwhile, with the presence of Sa-16S rDNA, the auxiliary probes and primers stimulated the SDA reaction under the action of Klenow fragment (3'-5' exo-) and Nt. BbvCI specifically recognized Sa-16S rDNA to form a defective T-junction structure and generated second primers to initiate the cycles. Such a structure transformed the input signal (Sa-16S rDNA) into substantial single-stranded DNA products (SP) through SDA. SP acted as activators and activated arbitrary side chain cleavage of CRISPR/Cas12a (trans-cleavage) and further realized effective annihilation of ECL signals. This ECL platform demonstrated desirable assay performance for Sa-16S rDNA with a wide response range of 1 fM to 10 nM, and the limit of detection was 0.437 fM (S/N = 3), showing good sensitivity and specificity. Therefore, the method not only expanded the applications of CRISPR/Cas12a but also opened up a novel strategy for clinical diagnosis.

Colorimetric and Raman dual-mode lateral flow immunoassay detection of SARS-CoV-2N protein antibody based on Ag nanoparticles with ultrathin Au shell assembled onto Fe3O4 nanoparticles.

Serological antibody tests are useful complements of nuclei acid detection for SARS-CoV-2 diagnosis, which can significantly improve diagnostic accuracy. However, antibody detection in serum or plasma remains challenging to do with high sensitivity. In this study, Ag nanoparticles with ultra-thin Au shells embedded with 4-mercaptobenzoic acid (MBA) (AgMBA@Au) were manufactured and then assembled onto Fe3O4 surface by electrostatic interaction to construct the Fe3O4-AgMBA@Au nanoparticles (NPs) with magnetic-Raman-colorimetric properties. Based on the composite nanoparticles, a colorimetric and Raman dual-mode lateral flow immunoassay (LFIA) for ultrasensitive identification of SARS-CoV-2 nucleocapsid (N) protein antibody was constructed. The magnetic nanoparticles (Fe3O4 NPs) were acted as the core and coated a layer of AgMBA@Au particles on the surface by electrostatic interaction to prepare Fe3O4-AgMBA@Au NPs, which can amplify the SERS signal due to multiple AgMBA@Au particles concentrated on a single magnetic nanoparticle. Moreover, the Fe3O4-AgMBA@Au NPs facilitated pre-purifying sample using magnetic separation, and complex matrix interference would be greatly decreased in the detection. The Fe3O4-AgMBA@Au NPs modified with N protein recognized and bound with N protein antibodies, which were trapped on the T-line, forming color band for observing detection. Under optimal conditions, the N protein antibodies could be qualitatively detected in colorimetric mode with the visual limit of 10-8mg/mL and quantitatively detected by SERS signals between 10-6 and 10-10mg /mL with 0.08pg/mL detection limit. The coefficients variations (CV) of intra-assay was 8.0%, whereas of inter-assay was 11.7%, confirming of good reproducibility. Finally, this approach was able to discriminate between positive, negative, and weakly positive samples when detecting 107 clinical serum samples. The process enables highly sensitive quantitative assays that are valuable for evaluating disease processes and guiding treatment. Colorimetric and Raman dual-mode LFIA detection of SARS-CoV-2N protein antibody based on Fe3O4-AgMBA@Au nanoparticles.

Evaluation of a direct phage DNA detection-based Taqman qPCR methodology for quantification of phage and its application in rapid ultrasensitive identification of Acinetobacter baumannii

BackgroundRapid phage enumeration/quantitation and viable bacteria determination is critical for phage application and treatment of infectious patients caused by the pathogenic bacteria.MethodsIn the current study, a direct phage DNA detection-based Taqman qPCR methodology for quantification of phage P53 and rapid ultrasensitive identification of Acinetobacter baumannii (A. baumannii) was evaluated.ResultsThe assay was capable of quantifying P53 phage DNA without DNA extraction and the detection limit of the assay was 550 PFU/mL. The agreement bias between the quantitative results of three different phage concentrations in this assay and double agar overlay plaque assay were under 3.38%. Through the built detection system, down to 1 log CFU/mL of viable A. baumannii can be detected within 4 h in A. baumannii spiked swab and bronchoalveolar lavage fluid samples. Compared with the Taqman qPCR that targets the conserved sequence of A. baumannii, the sensitivity of the assay built in this study could increase four orders of magnitude.ConclusionsThe methodology offers a valid alternative for enumeration of freshly prepared phage solution and diagnosis of bacterial infection caused by A. baumannii or other bacterial infection in complicated samples through switching to phages against other bacteria. Furthermore, the assay could offer drug adjustment strategy timely owing to the detection of bacteria vitality.

Ultrasensitive surface‐enhanced Raman spectroscopy detection of explosive molecules with multibranched silver nanostructures

AbstractMultibranched silver nanostructures (AgNSts) have attracted much attention as promising candidates for surface‐enhanced Raman spectroscopy (SERS) due to their unique plasmonic properties. We have chemically synthesized silver nanodendrites (AgNDs) and silver stars (AgSs) and investigated their SERS performance for trace‐level molecule detection. High enhancement factor (EF) ~1010 and attomolar detection limits were obtained with multibranched nanostructure‐based SERS substrates for methylene blue, thiram, and phosmet. The confinement of the local fields at the sharp tips and in the intrabranch and interbranch nanogaps of AgNDs offer highly dense three‐dimensional (3D) SERS “hot spots” and large signal enhancements of ~109. Further, the as‐prepared AgND‐based substrates were utilized for the ultrasensitive identification of explosive molecules 2,4‐DNT, PNBA, and PA with limit‐of‐detection (LOD) down to ~5.3 × 10−16, 2.9 × 10−16, and 3.8 × 10−12 M concentrations, respectively. The spectral modifications and appearance of new SERS peaks in the low wavenumber region indicate the metal–molecule complex formation and confirm the contribution of the chemical enhancement mechanism. The narrow spectral widths of Raman peaks allow the highly selective multiplexed detection of explosive molecules from the two‐component (2‐plex) mixture of dyes with different concentrations. Further, the density functional theory (DFT)‐based Raman spectrum calculations of the molecules exhibit a reasonably good correspondence with the experimental results. Therefore, the molecularly specific and distinguishably sharp Raman features enable the ultrasensitive and multiplexed detection of analytes molecules with our AgNSt‐based SERS substrates.

Ultrasensitive luminescent turn-on detection of perchlorate particulates by triggering supramolecular self-assembly of platinum(II) complex in hydrogel matrix

Onsite ultrasensitive identification of the airborne or deposited perchlorate particulates is crucial for homeland security, forensic analysis, and environment monitoring. However, few of the currently developed luminescent sensor focus on the direct and ultratrace detection of its solid particulates as the low level of analyte and the difficulty in exhaustive collection of the trace sample. Here, a general design strategy for the direct, ultrasensitive, and specific luminescent turn-on sensing of perchlorate particulates is proposed by triggering supramolecular self-assembly of cationic platinum(II) complex in a hydrogel matrix, in which the self-assembly of platinum(II) complex endows the luminescence turn-on response and the hydrogel matrix creates excellent confinement effect for terminating the diffusion of generated luminescent 1D aggregates. The proposed hydrogel-based sensor is capable to detect the airborne perchlorate particulate of 1.12 ng by naked eye and 0.11 fg by common optical microscope. Further practical sensing performances for real firecracker and buried perchlorate in soil demonstrates the excellent environmental adaptability, and the latent fingerprint visualization shows great potential for clue tracking. This study provides an inspirable strategy for designing ultrasensitive luminescent sensor for trace solid sample.

Surface engineering of roselike lanthanum molybdate electrocatalyst modified screen-printed carbon electrode for robust and highly sensitive sensing of antibiotic drug

Creation of efficient electrochemical sensor for the ultrasensitive identification of food toxic chemicals (organo arsenic compound). We also investigated the possibility of rapid identification of coccidiostat using rose-like tetragonal lanthanum molybdates as a modified electrode. Moreover, the lanthanum molybdates modified electrodes were concentrated on the evolution of electrochemical reduction achievements for the analysis of organo arsenic compound roxarsone (ROX) in different meat products. The modified sensor is constructed by rose-like lanthanum molybdates (RL-La2(MoO4)3) on to an SPCE (screen-printed carbon electrode). The two-dimensional nanosheets like RL-La2(MoO4)3 electrocatalyst reveal enhanced detection towards ROX by the combination of electrostatic attraction between ROX and RL-La2(MoO4)3 surfaces. The modified sensor has exhibited a very low detection limit (12.4 nM) in the neutral supporting electrolyte by electrochemical methods. Furthermore, the modified sensor ranges of ROX is 0.025–2650 μM. Also, sensitivity of RL-La2(MoO4)3 electrode is calculated as 13.366 μA·μM−1·cm−2 and 42.619 μA·μM−1·cm−2. The lanthanum molybdates modified device shows good stability and reasonable reproducibility. The obtained electrochemical results are satisfactory based on the RL-La2(MoO4)3 electrocatalyst modified sensor for food analysis.

A high-throughput and ultrasensitive identification methodology for unauthorized GMP component based on suspension array and logical calculator

To solve the problem of the unauthorized GMP components within import and export goods, the LI-US (Logic Identification of unauthorized GMP content by Universal-primer Suspension-array) system, which takes advantage of suspension array and logic calculator, was developed in the present study. Seventeen signal input channels have been optimized and validated in our research to ensure the multiplex practicality of the LI-US system. Three LI-US logic gates, including a YES gate, an OR gate and an AND gate, were designed as different detection strategies for GMP identification. The feasibility and specificity of the LI-US system were validated in the present study. Combining the optimization and evaluation of the signal input procedure, the sensitivity of this LI-US system reached 0.05% of the GMP mass concentration. The practicability evaluation of LI-US demonstrated its application within different substrates and varieties. In conclusion, the LI-US system was developed with extremely high specificity, sensitivity and practicability among different substrates and varieties, which could meet the demands of unauthorized GMP contents for both import and export goods.

Magnetic nanochain integrated microfluidic biochips

Microfluidic biochips hold great potential for liquid analysis in biomedical research and clinical diagnosis. However, the lack of integrated on-chip liquid mixing, bioseparation and signal transduction presents a major challenge in achieving rapid, ultrasensitive bioanalysis in simple microfluidic configurations. Here we report magnetic nanochain integrated microfluidic chip built upon the synergistic functions of the nanochains as nanoscale stir bars for rapid liquid mixing and as capturing agents for specific bioseparation. The use of magnetic nanochains enables a simple planar design of the microchip consisting of flat channels free of common built-in components, such as liquid mixers and surface-anchored sensing elements. The microfluidic assay, using surface-enhanced Raman scattering nanoprobes for signal transduction, allows for streamlined parallel analysis of multiple specimens with greatly improved assay kinetics and delivers ultrasensitive identification and quantification of a panel of cancer protein biomarkers and bacterial species in 1 μl of body fluids within 8 min.

Ultra-precise detection of mutations by droplet-based amplification of circularized DNA

BackgroundNGS (next generation sequencing) has been widely used in studies of biological processes, ranging from microbial evolution to cancer genomics. However, the error rate of NGS (0.1 % ~ 1 %) is still remaining a great challenge for comprehensively investigating the low frequency variations, and the current solution methods have suffered severe amplification bias or low efficiency.ResultsWe creatively developed Droplet-CirSeq for relatively efficient, low-bias and ultra-sensitive identification of variations by combining millions of picoliter uniform-sized droplets with Cir-seq. Droplet-CirSeq is entitled with an incredibly low error rate of 3 ~ 5 X 10-6. To systematically evaluate the performances of amplification uniformity and capability of mutation identification for Droplet-CirSeq, we took the mixtures of two E. coli strains as specific instances to simulate the circumstances of mutations with different frequencies. Compared with Cir-seq, the coefficient of variance of read depth for Droplet-CirSeq was 10 times less (p = 2.6 X 10-3), and the identified allele frequency presented more concentrated to the authentic frequency of mixtures (p = 4.8 X 10-3), illustrating a significant improvement of amplification bias and accuracy in allele frequency determination. Additionally, Droplet-CirSeq detected 2.5 times genuine SNPs (p < 0.001), achieved a 2.8 times lower false positive rate (p < 0.05) and a 1.5 times lower false negative rate (p < 0.001), in the case of a 3 pg DNA input. Intriguingly, the false positive sites predominantly represented in two types of base substitutions (G- > A, C- > T). Our findings indicated that 30 pg DNA input accommodated in 5 ~ 10 million droplets resulted in maximal detection of authentic mutations compared to 3 pg (p = 1.2 X 10-8) and 300 pg input (p = 2.2 X 10-3).ConclusionsWe developed a method namely Droplet-CirSeq to significantly improve the amplification bias, which presents obvious superiority over the currently prevalent methods in exploitation of ultra-low frequency mutations. Droplet-CirSeq would be promisingly used in the identification of low frequency mutations initiated from extremely low input DNA, such as DNA of uncultured microorganisms, captured DNA of target region, circulation DNA of plasma et al, and its creative conception of rolling circle amplification in droplets would also be used in other low input DNA amplification fields.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-2480-1) contains supplementary material, which is available to authorized users.

Toward Non-Enzymatic Ultrasensitive Identification of Single Nucleotide Polymorphisms by Optical Methods

Single nucleotide polymorphisms (SNPs) are single nucleotide variations which comprise the most wide spread source of genetic diversity in the genome. Currently, SNPs serve as markers for genetic predispositions, clinically evident disorders and diverse drug responses. Present SNP diagnostics are primarily based on enzymatic reactions in different formats including sequencing, polymerase-chain reaction (PCR) and microarrays. In these assays, the enzymes are applied to address the required sensitivity and specificity when detecting SNP. On the other hand, the development of enzyme-free, simple and robust SNP sensing methods is in a constant focus in research and industry as such assays allow rapid and reproducible SNP diagnostics without the need for expensive equipment and reagents. An ideal method for detection of SNP would entail mixing a DNA or RNA target with a probe to directly obtain a signal. Current assays are still not fulfilling these requirements, although remarkable progress has been achieved in recent years. In this review, current SNP sensing approaches are described with a main focus on recently introduced direct, enzyme-free and ultrasensitive SNP sensing by optical methods.