Label-free Strategy Research Articles

Terminal Deoxynucleotidyl Transferase and Rolling Circle Amplification Induced G-triplex Formation: A Label-free Fluorescent Strategy for DNA Methyltransferase Activity Assay

It is of great significance to sensitively detect DNA methyltransferase (MTase) activity and screen its inhibitory drugs in the field of human cancer diagnosis and disease therapeutics. In this work, based on terminal deoxynucleotidyl transferase (TdT)-induced rolling circle amplification (RCA), we construct a label-free and highly sensitive fluorescence biosensing strategy for DNA MTase activity analysis. The hairpin substrate is methylated by Dam MTase and cleaved by Dpnl to generate a DNA fragment with a free 3′-hydroxy (3′−OH) terminus that is extended by TdT to obtain a long adenine (poly-A) tail. Subsequently, the resulting poly-A work as primer triggers RCA reaction and generate lots of G-triplexes which can combine with Thioflavin T (ThT) to achieve the apparent fluorescence signal. Integrate TdT-induced RCA with G-triplex@ThT, Dam MTase can be detected down to 0.058 U/mL with a wide linear range from 0.1 U/mL to 40 U/mL. In addition, the method exhibits high selectivity to other MTases. We also successfully demonstrate this work can be used in complex matrixes assay and screen MTase inhibitors. Thus, in the area of DNA MTase-related cancer diagnosis and disease therapeutics, the developed method may be a promising tool for the detection of Dam MTase.

An exonuclease-assisted fluorescence sensor for assaying alkaline phosphatase based on SYBR Green I

In this report, we propose a fast, reliable and convenient approach to determine the alkaline phosphatase (ALP) activity based on a label-free fluorescence strategy. Upon catalysis of ALP, dephosphorylated dsDNA hampers the λ exonuclease (λexo) cleavage, shows high affinity to SYBR Green I (SG I), resulting in a strong fluorescence emission peak at 520 nm. In the absence of ALP, the dsDNA with 5′-phosphoryl-termini could be employed as a substrate of λexo. After cleavage, a weak fluorescence emission peaks at 520 nm could be observed. The assay was both selective and sensitive, and the detection limit was found to be as low as 3 U/L. This method was utilized to evaluate Na3VO4 as ALP inhibitor. The method was successfully applied to the determination of the activity of ALP in spiked human serum samples.

Flexible Biosensors for the Impedimetric Detection of Protein Targets Using Silk-Conductive Polymer Biocomposites.

To expand the applications of flexible biosensors in point-of-care healthcare applications beyond monitoring of biophysical parameters, it is important to devise strategies for the detection of various proteins and biomarkers. Here, we demonstrate a flexible, fully organic, biodegradable, label-free impedimetric biosensor for the critical biomarker, vascular endothelial growth factor (VEGF). This biosensor was constructed by photolithographically patterning a conducting ink consisting of a photoreactive silk sericin coupled with a conducting polymer. These functional electrodes are printed on flexible fibroin substrates that are controllably thick and can be free-standing, or conform to soft surfaces. Detection was accomplished via the antibody to VEGF which was immobilized within the conducting matrix. The results indicated that the developed flexible biosensor was highly sensitive and selective to the target protein, even in challenging biofluids such as human serum. The biosensors themselves are biocompatible and degradable. Through this work, the developed flexible biosensor based on a simple and label-free strategy can find practical applications in the monitoring of wound healing or early disease diagnosis.

Neuropeptidomics: Comparison of parallel reaction monitoring and data-independent acquisition for the analysis of neuropeptides using high-resolution mass spectrometry.

Targeted peptide quantitation by mass spectrometry is a rapidly emerging field. Traditionally it relied on the development and validation of multiple reaction monitoring assays that could comply with a high level of sensitivity, specificity, accuracy and reproducibility in complex biological samples. However, with the introduction of high-resolution mass spectrometers, other acquisition modes could provide more comprehensive datasets for identification and quantification but also for in-depth data mining. The objective of this study was to evaluate two analytical approaches, parallel-reaction monitoring (PRM) and data-independent analysis (DIA) using a hybrid Quadrupole-Orbitrap mass spectrometer for the quantification of neuropeptides in animal spinal cord tissues. Mouse spinal cord tissues were harvested, homogenized and neuropeptides extracted using a C18 solid-phase extraction protocol. Chromatography was achieved using a Thermo Biobasic C8 100 × 1 mm (5 μm) column. The initial mobile phase conditions consisted of acetonitrile and water (both containing 0.1% of formic acid) at a ratio of 5:95. An 11 min linear gradient was applied up to a ratio of 50:50 and maintained for 3 min. The flow rate was fixed at 75 μL/min and 2 μL of sample was injected. Mass spectrometry analyses were performed using a Thermo Q Exactive Plus MS using PRM and DIA approaches. Quantitative data using an isotopic dilution and a label-free strategy were obtained for both methods and statistically compared. Using both approaches, we were able to clearly detect endogenous neuropeptides. However, with DIA, mass spectra alone could not distinguish Leu-Enk and Met-Enk. We used a Bland-Altman plot (Difference plot) to analyze the agreement between both approaches and no systematic bias was observed. Further statistical analyses, including variance analysis, showed more variability in DIA compared with PRM mode. Further analyses were performed using a label-free approach and confirmed an increase of the variance using a DIA approach.

Direct detection of label-free blood fingermarks by SECM imaging

A label-free strategy for imaging blood fingermarks (BFMs) on a hydrophobic poly(vinylidene difluoride) (PVDF) membrane has been developed using scanning electrochemical microscopy (SECM) in the surface interrogation (SI) mode. In this approach, hexaammineruthenium chloride ([Ru(NH3)6]Cl3) was chosen as a redox mediator to react chemically with the electroactive hemichrome (HC) present in the BFM ridge deposits but not in the furrow regions. This provides a sharp contrast of the current response at the tip of the electrode between ridges and furrows and results in an electrochemical image of the BFM with the high resolution required for personal identification. In addition, this approach has high sensitivity, with the ability to directly image latent BFMs deposited with 1000-fold diluted blood on PVDF membranes. Such marks cannot be detected by optical microscopy. Moreover, for the first time a commercial PVDF membrane was used instead of adhesive forensic tape to lift the BFMs from common glass and multicolored banknote surfaces, and the lifted BFMs could also be efficiently imaged by this electrochemical method.

A Multiplex Fragment-Ion-Based Method for Accurate Proteome Quantification.

Multiplex proteome quantification with high accuracy is urgently required to achieve a comprehensive understanding of dynamic cellular and physiological processes. Among the existing quantification strategies, fragment-ion-based methods can provide highly accurate results, but the multiplex capacity is limited to 3-plex. Herein, we developed a multiplex pseudo-isobaric dimethyl labeling (m-pIDL) method to extend the capacity of the fragment-ion-based method to 6-plex by one-step dimethyl labeling with several millidalton and dalton mass differences between precursor ions and enlarging the isolation window of precursor ions to 10 m/ z during data acquisition. m-pIDL showed high quantification accuracy within the 20-fold dynamic range. Notably, the ratio compression was 1.13-fold in a benchmark two-proteome model (5:1 mixed E. coli proteins with HeLa proteins as interference), indicating that by m-pIDL, the ratio distortion of isobaric labeling approaches and the approximate 40% ratio shift of the label-free quantification strategy could be effectively eliminated. Additionally, m-pIDL did not show ratio variation among post-translational modifications (CV = 6.66%), which could benefit the measurement of universal protein properties for proteomic atlases. We further employed m-pIDL to monitor the time-resolved responses of the TGF-β-induced epithelial-mesenchymal transition (EMT) in lung adenocarcinoma A549 cell lines, which facilitated the finding of new potential regulatory proteins. Therefore, the 6-plex quantification of m-pIDL with the remarkably high accuracy might create new prospects for comprehensive proteome analysis.

DNA three-way junction-actuated strand displacement for miRNA detection using a fluorescence light-up Ag nanocluster probe.

A rapid and label-free fluorescence biosensing strategy for highly sensitive detection of microRNA-122 (miR-122) has been developed by the combination of DNA three-way junction (TWJ)-actuated strand displacement and a fluorescence light-up Ag nanocluster (AgNC) probe. In the presence of target miR-122, the attachment of miR-122 to its complementary DNA results in the unblocking of the toehold and branch migration domains in the TWJ, activating the strand displacement reaction (SDR) accompanied by the proximity between the G-rich DNA probe and DNA-AgNC probe; thus a remarkably enhanced fluorescence signal of AgNCs can be obtained owing to the G-rich fluorescence enhancement mechanism. The results reveal that this biosensor exhibits superb specificity and high sensitivity toward miR-122 with a detection limit of 0.030 nM. In addition, the practicality of the biosensor is demonstrated by analyzing miR-122 in three cell lines with satisfactory results. Furthermore, by the utilization of the toehold-mediated SDR and DNA-AgNC conjugates, this proposed strategy offers the advantages of rapidness, convenience, low cost, and simplified operation without the need for biological labeling and the addition of enzymes. Thus, the constructed biosensor might provide a valuable and practical tool for detecting miRNA and the related clinical diagnosis and fundamental biomedicine research.

Aptamer recognition-trigged label-free homogeneous electrochemical strategy for an ultrasensitive cancer-derived exosome assay.

We developed an aptamer recognition-trigged label-free homogeneous electrochemical biosensing method for highly selective and ultrasensitive detection of cancer-derived exosomes.

Visualization of the intracellular location and stability of DNA flowers with a label-free fluorescent probe.

Rolling circle amplification (RCA) and rolling circle transcription (RCT) can be used to fabricate various structures and organize functional materials for biological applications. The full understanding of the interactions between RCA/RCT-derived structures and live cells is urgently demanded. Here, we present a label-free fluorescent strategy to study the intracellular location and stability of RCA-based DNA flowers in live cells. The DNA flower structures are co-assembled with carbazole-based biscyanine fluorophores, which are DNA detecting molecules and characterized by restriction of intramolecular rotation (RIR) induced strong fluorescent emission. When biscyanine molecules are encapsulated in the DNA flowers via electrostatic attraction, these confined RIR dyes can produce strong luminescent emission. Using this advantage, we use the RIR enhanced technique for direct visualization of the distribution and degradation of DNA flowers in live cellular systems. Our current research could be adapted to other advanced DNA-based materials, providing a new strategy to fabricate fluorescent DNA materials and realize controllable release of payloads.

A label-free colorimetric strategy for facile and low-cost sensing of ascorbic acid using MnO2 nanosheets

A facile and sensitive colorimetric method for determining ascorbic acid (AA) was established using MnO2 nanosheets. Addition of AA to the sensor system caused the transformation of solution color from brown to colorless.

Label-Free Electrochemical Strategy for Probing TdT and Exo I Activity by Silver-Based Square Wave Voltammetry

Monitoring DNA-related enzymes activity precisely and reliably is absolutely vital to better grasp their roles in the initiation and progression of cancer, thus further exploring their functions in cancer diagnosis and treat. Here, we establish a label-free electrochemical biosensor for investigating both terminal deoxynucleotidyl transferase (TdT) and exonuclease I (Exo I) activity by silver-based square wave stripping voltammetry (SWV). The strategy is achieved by two attractive points in design: a TdT-derived enzymatic synthesis of randomly arrayed C-rich DNA sequence and an Exo I-guided enzymatic degradation. This C-rich DNA-templated silver nanoclusters (AgNCs) serve as the electrochemical signal probe, and a signal on-off modulated process is displayed for the existence of TdT and Exo I, respectively. On account of AgNCs-based square wave stripping voltammetry, testing TdT has a linear concentration range of 0.001 to 5 U/mL with a detection limit of 0.00044 U/mL (S/N = 3). Additionally, Exo I with a detection limit of 0.027 U/mL (S/N = 3) can be analyzed with a linear range of 0.1 to 500 U/mL. The real application of the proposed method in their inhibitors screening is also discussed well. The biosensor provides a powerful idea for the ultrasensitive bio-analysis and shows broad clinical application prospect.

Engineering Nanozymes Using DNA for Catalytic Regulation.

DNA treatment of metal nanoparticles provides a potent tool for tuning their native properties and constructing advanced materials. However, there have been limited studies on interactions between DNA and nanomaterial-based artificial enzymes (nanozymes) to influence their intrinsic peroxidase-like properties. Here, we present the utilization of DNA as a capping ligand to engineer various bio-nanointerfaces for high-precise and adjustable regulation of catalytic behaviors of nanozymes toward the oxidation of substrates. The treatment of stiff double-stranded DNA only induced a negligible enhancement of the catalytic activity of nanozymes, and both coil-like single-stranded DNA and hairpin DNA-capped nanoparticles produced a medium signal increase. Interestingly, hybridization chain reaction (HCR) product-treated nanoparticles showed the highest peroxidase-like activities among four DNA structures. Furthermore, significant parameters that influence HCR process and the modulation of catalysis, such as the concentration of the hairpin DNA, the ionic strength, and the amount of nanozyme, were also systematically investigated. On the basis of HCR amplification and iron oxide (Fe3O4) nanoparticles, we develop a simple, fast, label-free, and sensitive colorimetric strategy for sensing of a Yersinia pestis-relevant DNA sequence with a detection limit as low as 100 pM as well as single nucleotide polymorphism discrimination. These results highlight DNA engineering as a facile strategy to regulate the catalytic activities of nanozymes and understand the interactions between metallic nanoparticles and nucleic acids for biosensing applications.

Proteomics of Bulked Rachides Combined with Documented QTL Uncovers Genotype Nonspecific Players of the Fusarium Head Blight Responses in Wheat.

Fusarium head blight (FHB) is a destructive disease of wheat that reduces yield and grain quality. High-throughput proteomic techniques have been used to identify a wide range of candidate proteins involved in host resistance. The majority of the published works on the proteomics of the wheat response to Fusarium graminearum infection are case specific. In the current study, a high-throughput quantitative label-free strategy was employed on bulked rachides of F. graminearum-infected wheat collected from multiple genotypes. Differentially accumulated proteins among the following four pools were identified: mock-inoculated FHB-resistant accessions (RM), mock-inoculated FHB-susceptible accessions (SM), F. graminearum-inoculated FHB-resistant accessions (RFg), and F. graminearum-inoculated FHB-susceptible accessions (SFg). Four pairs of comparisons were made: RFg versus RM, SFg versus SM, RM versus SM, and RFg versus SFg. Proteins were projected onto the consensus intervals of previously reported quantitative trait loci in the FHB-resistant pool by blasting against the Chinese Spring reference sequences. In addition to proteins previously reported in the host response to Fusarium spp., new candidates have emerged in association with resistance or susceptibility, including a group 3 late embryogenesis abundant as a resistance-related protein and a purple acid phosphatase as a susceptibility protein. The protein atlas presented here provides new perspectives on the interaction between F. graminearum and wheat.

Ultrahigh Selective Colorimetric Quantification of Chromium(VI) Ions Based on Gold Amalgam Catalyst Oxidoreductase-like Activity in Water.

Hexavalent chromium ion (Cr6+) is one of the most toxic substances for plants, for animals, and is a confirmed human respiratory carcinogen. However, so far, there are few independent and efficient colorimetric methods for detection of Cr6+. Here, we introduce a convenient, label-free, catalysis-based, and efficient strategy for quantification of Cr6+ by using a colorimetric sensing probe 3,3',5,5'-tetramethylbenzidine (TMB). In the presence of a trace amount of gold amalgam nanocomposites (Au@Hg) and Cr6+, TMB can be oxidized to oxTMB and the color changed to an intense blue that was observed by naked-eye and absorption spectroscopic method. In addition, the colorimetric method shows the high selectivity against 34 other interfering substances, and it can be performed at room temperature, in water, and requires only ∼5 min. Thus, the catalysis-based colorimetric assay for accurate and ultrahigh selective identification of Cr6+ will find widespread use in the world.

Molecularly imprinted nanoparticles based plasmonic sensors for real-time Enterococcus faecalis detection

Human fecal contamination poses a crucial environmental and health threat in recent years, resulting in the difficulties of access to clean water. According to the World Health Organization, several fecal bacteria, particularly Enterococci species, are present in human intestinal flora. Enterococcus faecalis (E. faecalis) is one of the indicator bacteria that have been utilized as a pollution indicator in water. However, existing technologies and detection strategies face multiple challenges in terms of low affinity for detection and labelling requirements that limit their access to large scale applications. Here, we present a label-free molecular fingerprinting strategy on a plasmonic sensor to detect E. fecalis from aqueous and seawater samples. The kinetic performance of platform was comprehensively evaluated and the platform provided four orders of magnitude detection range with a low limit of detection (down to ~100 bacteria/mL) and a high correlation coefficient value (> 0.99) in the range of 2 × 104–1 × 108 cfu/mL. The platform also denoted a selectivity and specificity while other bacteria (E. coli, B. subtilis, and S. aureus) samples were applied. Multiple time use and relatively long shelf-life are superior to the existing modality. The presented method is one of the fascinating surface modification technique that utilizes biotarget as a recognition element itself, providing a broad range of versatility to replica other biotargets with different molecular structure, size, and physicochemical properties. Such a reliable and versatile platform would hold potential applications from microbiome characterization to forensics by revitalizing obsolescent detection strategies.

Investigation on rat intestinal homeostasis alterations induced by 7-day simulated microgravity effect based on a proteomic approach

The present study aims at investigating alterations of rat intestinal homeostasis induced by 7-day simulated microgravity (SMG) effect through a proteomic approach. Tail-suspension model was used to simulate microgravity effect and a label-free quantitative proteomic strategy was employed to determine proteins in rat intestine. As a result, 717 differently expressed proteins were identified and 29 proteins were down-regulated while 688 proteins were up-regulated. 283 out of 717 proteins were categorized into 7 clusters with DAVID (version 6.8). The three highest enrichment scores were annotation cluster I about cell-cell adhesion (46 proteins with enrichment score of 16.67), annotation cluster II about carbohydrate metabolism (32 proteins with enrichment score of 6.17) and annotation cluster III about activity of pepdtidase (17 proteins with enrichment score of 4.74). Results of rat intestine proteomics indicate that SMG might disrupt intestinal homeostasis, which possibly resulted in opening of intestinal epithelial barrier (IEB), potentially leading to risk of systemic inflammatory response (SIR) and inflammatory bowel diseases (IBD). The present results also provide some useful information for mechanism and countermeasures of intestine injuries induced by microgravity.

A simple enzyme-assisted cascade amplification strategy for ultrasensitive and label-free detection of DNA.

A simple fluorescence biosensor is developed based on the enzyme-assisted cascade amplification strategy. The amplification system consists of a hairpin-structure DNA (H-DNA) and exonuclease III. The target DNA can hybridize with the H-DNA and initiate exonuclease III-assisted target recycling amplification to generate abundant G-rich DNA (G-DNA). One region of G-DNA is designed to possess the same sequence as target DNA. Thus, the G-DNA can also hybridize with H-DNA and initiate the digestion of H-DNA. The cascade strategy in this amplification system causes the concentration of G-DNA to grow exponentially. The fluorescence intensity of N-methylmesoporphyrin IX (NMM) is highly enhanced due to the formation of G-quadruplex configuration. Under optimal conditions, the cascade system could achieve an admirable sensitivity with a detection limit of 52fM for HIV DNA, and guarantees a satisfactory specificity. Moreover, the cascade system could be implemented for other target DNA detections by substituting the recognition region of the H-DNA. In this way, a detection limit of 65fM for HBV DNA could be achieved by the cascade system. The target DNA analysis in a real serum sample further indicates that this biosensor has potential for future application in clinical diagnosis. Graphical abstract A simple and label-free cascade amplification strategy is developed by exploiting hairpin DNA and EXO III for sensitive DNA detection.

Photoelectrochemical Approach to Apoptosis Evaluation via Multi-Functional Peptide- and Electrostatic Attraction-Guided Excitonic Response.

The excitonic response between nanomaterials is distance-dependent, and thus, interparticle distance is a key factor in fabricating diverse photoelectrochemical (PEC) systems. Current studies focus on DNA-mediated regulation of interparticle distance. However, limited by high demands of base-pairing and flexibility of DNA, it is hard for DNA to achieve precise regulation, especially in a short distance. To pursue better PEC performances in bioanalyses, alternative biological materials should be explored to replace DNA as new "distance controllers". In this work, a peptide with three functional sequences is designed to control interparticle distance between positive-charged Au nanoparticles ((+) AuNPs) and negative-charged CdTe quantum dots ((-) CdTe QDs). Relying on the function of binding sequence, (+) AuNPs and (-) CdTe QDs may be separated to a certain distance by the multifunctional peptide. In this case, the excitonic response is relatively weak, and an evident PEC response can be observed. Because it contains the substrate sequence of caspase-3, the peptide is cleaved in the presence of caspase-3. As a result, without the support of intact peptide, electrostatic attraction plays a dominant role, leading to the aggregation of oppositely charged AuNPs and CdTe QDs, which strengthens the excitonic response and attenuates the PEC response. On the basis of these principles, a novel PEC approach was fabricated to sensitively quantify caspase-3. Meanwhile, caspase-3 in staurosporine-treated A549 cells are also determined by the approach, and the obtained results agree well with the fluorescent intensity of confocal images, manifesting that the proposed PEC method can monitor apoptosis in a label-free strategy. Overall, the study reveals the capability of peptides in controlling interparticle distance of nanomaterials, which may accelerate the development of peptide-based PEC analytical methods.

Plasmonic bacteria on a nanoporous mirror via hydrodynamic trapping for rapid identification of waterborne pathogens

A rapid, precise method for identifying waterborne pathogens is critically needed for effective disinfection and better treatment. However, conventional methods, such as culture-based counting, generally suffer from slow detection times and low sensitivities. Here, we developed a rapid detection method for tracing waterborne pathogens by an innovative optofluidic platform, a plasmonic bacteria on a nanoporous mirror, that allows effective hydrodynamic cell trapping, enrichment of pathogens, and optical signal amplifications. We designed and simulated the integrated optofluidic platform to maximize the enrichment of the bacteria and to align bacteria on the nanopores and plasmonic mirror via hydrodynamic cell trapping. Gold nanoparticles are self-assembled to form antenna arrays on the surface of bacteria, such as Escherichia coli and Pseudomonas aeruginosa, by replacing citrate with hydroxylamine hydrochloride in order to amplify the signal of the plasmonic optical array. Owing to the synergistic contributions of focused light via the nanopore geometry, self-assembled nanoplasmonic optical antennas on the surface of bacteria, and plasmonic mirror, we obtain a sensitivity of detecting E. coli as low as 102 cells/ml via surface-enhanced Raman spectroscopy. We believe that our label-free strategy via an integrated optofluidic platform will pave the way for the rapid, precise identification of various pathogens.

Liquid crystals-based sensor for the detection of lithocholic acid coupled with competitive host-guest inclusion.

A simple, low-cost and label-free strategy for detecting lithocholic acid (LCA) was designed at the liquid crystals (LCs)/aqueous interface via competitive host-guest inclusion. In this method, sodium dodecyl sulfate (SDS) was initially adsorbed on the fluid interface and induced LCs to adopt the homeotropic ordering. Inclusion complexation of SDS and β-cyclodextrin (β-CD) disturbed interaction between LCs and SDS and evoked LCs to keep a tilted alignment. When injecting LCA into the mixed solution of SDS and β-CD, SDS excluded from the cavity of β-CD by competitive host-guest inclusion and could be re-adsorbed at the LCs/aqueous interface, resulting in the orientational transition of LCs from tilted to homeotropic state. Correspondingly, a bright-to-dark optical response was observed under polarized optical microscope (POM). The as-prepared LCs-based sensor could detect LCA as low as about 2 μM in aqueous solution. Moreover, the practicability of the approach was validated by monitoring the known amount of LCA in human urine. This work offers an appealing approach for the detection of LCA which has a great potentiality in clinical diagnosis.