Label-free Strategy Research Articles

Rapid and label-free strategy for the sensitive detection of Hg2+ based on target-triggered exponential strand displacement amplification

A rapid and label-free strategy for sensitive detection of Hg2+ is developed based on target-triggered exponential strand displacement amplification (eSDA).

Single-probe multistate detection of DNA via aggregation-induced emission on a graphene oxide platform.

A simple, rapid method of fluorescent measurement of DNA hybridization in the presence of graphene (oxide) is presented. Conventional fluorescent dyes offer high performance in biosensors. However, labeling procedures are synthetically demanding in time and resources making it less cost-effective. Molecules with aggregation-induced-emission (AIE) property have advantages over traditional fluorescent molecules because of their intrinsic preference for detection as a turn-on probe and their single-molecule detection ability. Previous work has shown AIE dyes act as excellent "label-free" bioprobes with high sensitivity but with limited selectivity. Graphene oxide (GO) with its unique optical properties and affinity to different kinds of biomolecules can be used as an auxiliary to enhance selectivity of AIE dyes. In this work, we report a label-free strategy to detect DNA of particular sequence by water-soluble AIE probes with the aid of GO, supported by the computational explanations for this phenomenon.

A fluorescent aptasensing strategy for adenosine triphosphate detection using tris(bipyridine)ruthenium(II) complex containing six cyclodextrin units

A sensitive label-free fluorescent aptasensing strategy for the detection of adenosine triphosphate (ATP) has been developed with a metallocyclodextrin, tris(bipyridine)ruthenium(II) complex containing six cyclodextrin units (6CD-Ru), which exhibited much stronger emission signal compared to the parent compound Ru(bpy)3Cl2. Furthermore, the emission spectrum showed that the ATP-aptamer (ssDNA) could increase the fluorescence intensity of 6CD-Ru dramatically, attributed to the interaction between aptamer and cyclodextrin, which could provide protection to the ruthenium core from the quenching of emission by oxygen in the solution. With the addition of ATP, the interaction between aptamer and cyclodextrins on 6CD-Ru was diminished, since the ATP/aptamer complex had the priority to be formed, leading to the corresponding reduction of fluorescence intensity, which could be utilized to detect ATP quantitatively. A linear relationship was displayed between the fluorescence and the logarithm of ATP concentrations in the range from 1nmol/L to 1μmol/L with the detection limit of 0.5nmol/L (S/N=3). The proposed fluorescent aptasensing strategy exhibited high sensitivity and specificity, without any labeling or amplification procedures, and it could also be applied for the detection of many other aptamer-specific targets.

Polyphosphoric acid-induced perylene probe self-assembly and label-free fluorescence turn-on detection of alkaline phosphatase.

A label-free fluorescence turn-on strategy for alkaline phosphatase (ALP) detection was established based on its enzymatic catalyzed hydrolysis of polyphosphoric acid (PPA, an anionic polymer) that had been utilized for aggregation with our homemade positively charged perylene derivative (Probe-1) via noncovalent interactions. The disaggregation caused turn-on fluorescence signal which was recovered by the released Proble-1 molecules whose original strong fluorescence in an aqueous buffer solution had been quenched due to their previous aggregation induced by PPA. Such method presents its great advantages of free labeling, convenience and simplicity, cost effectiveness, high selectivity, and high sensitivity, with a detection limit of 0.5mU/mL of ALP. Graphical Abstract A label-free fluorescence turn-on strategy for alkaline phosphatase based on its enzymatic catalyzed hydrolysis of polyphosphoric acid that had been utilized for aggregation with our homemade positively charged perylene derivative via noncovalent interactions.

Evaluation of Spectral Counting for Relative Quantitation of Proteoforms in Top-Down Proteomics.

Spectral counting is a straightforward label-free quantitation strategy used in bottom-up proteomics workflows. The application of spectral counting in label-free top-down proteomics workflows can be similarly straightforward but has not been applied as widely as quantitation by chromatographic peak areas or peak intensities. In this study, we evaluate spectral counting for quantitative comparisons in label-free top-down proteomics workflows by comparison with chromatographic peak areas and intensities. We tested these quantitation approaches by spiking standard proteins into a complex protein background and comparing relative quantitation by spectral counts with normalized chromatographic peak areas and peak intensities from deconvoluted extracted ion chromatograms of the spiked proteins. Ratio estimates and statistical significance of differential abundance from each quantitation technique are evaluated against the expected ratios and each other. In this experiment, spectral counting was able to detect differential abundance of spiked proteins for expected ratios ≥2, with comparable or higher sensitivity than normalized areas and intensities. We also found that while ratio estimates using peak areas and intensities are usually more accurate, the spectral-counting-based estimates are not substantially worse. Following the evaluation and comparison of these label-free top-down quantitation strategies using spiked proteins, spectral counting, along with normalized chromatographic peak areas and intensities, were used to analyze the complex protein cargo of exosomes shed by myeloid-derived suppressor cells collected under high and low conditions of inflammation, revealing statistically significant differences in abundance for several proteoforms, including the active pro-inflammatory proteins S100A8 and S100A9.

Exposure of cultured fibroblasts to the peptide PR-11 for the identification of induced proteome alterations and discovery of novel potential ligands

The PR-11 peptide corresponds to the N-terminal and active region of the endogenously synthesized PR-39 molecule, of porcine origin. It is known to possess various biological effects including antimicrobial properties, angiogenic and anti-inflammatory activities. Apart from its reported activity as a proteasome inhibitor, a more comprehensive understanding of its function, at the molecular level, is still lacking. In this study, we used a label-free shotgun strategy to evaluate the proteomic alterations caused by exposure of cultured fibroblasts to the peptide PR-11. This approach revealed that more than half of the identified molecules were related to signalling, transcription and translation. Proteins directly associated to regulation of angiogenesis and interaction with the hypoxia-inducible factor 1-α (HIF-1α) were significantly altered. In addition, at least three differentially expressed molecules of the NF-κB pathway were detected, suggesting an anti-inflammatory property of PR-11. At last, we demonstrated novel potential ligands of PR-11, through its immobilization for affinity chromatography. Among the eluted molecules, gC1qR, a known complement receptor, appeared markedly enriched. This provided preliminary evidence of a PR-11 ligand possibly involved in the internalization of this peptide. Altogether, our findings contributed to a better understanding of the cellular pathways affected by PR-39 derived molecules.

High-throughput identification of telomere-binding ligands based on the fluorescence regulation of DNA-copper nanoparticles

Formation of the G-quadruplex in the human telomeric DNA is an effective way to inhibit telomerase activity. Therefore, screening ligands of G-quadruplex has potential applications in the treatment of cancer by inhibit telomerase activity. Although several techniques have been explored for screening of telomeric G-quadruplexes ligands, high-throughput screening method for fast screening telomere-binding ligands from the large compound library is still urgently needed. Herein, a label-free fluorescence strategy has been proposed for high-throughput screening telomere-binding ligands by using DNA-copper nanoparticles (DNA-CuNPs) as a signal probe. In the absence of ligands, human telomeric DNA (GDNA) hybridized with its complementary DNA (cDNA) to form double stranded DNA (dsDNA) which can act as an efficient template for the formation of DNA-CuNPs, leading to the high fluorescence of DNA-CuNPs. In the presence of ligands, GDNA folded into G-quadruplex. Single-strdanded cDNA does not support the formation of DNA-CuNP, resulting in low fluorescence of DNA-CuNPs. Therefore, telomere-binding ligands can be high-throughput screened by monitoring the change in the fluorescence of DNA-CuNPs. Thirteen traditional chinese medicines were screened. Circular dichroism (CD) measurements demonstrated that the selected ligands could induce single-stranded telomeric DNA to form G-quadruplex. The telomere repeat amplification protocol (TRAP) assay demonstrated that the selected ligands can effectively inhibit telomerase activity. Therefore, it offers a cost-effective, label-free and reliable high-throughput way to identify G-quadruplex ligands, which holds great potential in discovering telomerase-targeted anticancer drugs.

An enzyme-free surface plasmon resonance biosensing strategy for detection of DNA and small molecule based on nonlinear hybridization chain reaction

A label-free and enzyme-free surface plasmon resonance (SPR) biosensing strategy has been developed for highly sensitive and specific detection of target DNA by employing the nonlinear hybridization chain reaction (HCR) amplification. Nonlinear HCR is a hairpin-free system in which double-stranded DNA monomers could dendritically assemble into highly branched nanostructure upon introducing a trigger sequence. The target DNA partly hybridizes with capture probe on the gold sensing chip and the unpaired fragment of target DNA works as a trigger to initiate the nonlinear HCR, forming a chain-branching growth of DNA dendrimer by self-assembly. Real-time amplified SPR response is observed upon the introduction of nonlinear HCR system. The method is capable of detecting target DNA at the concentration down to 0.85 pM in 60min with a dynamic range from 1 pM to 1000 pM, and could discriminate target DNA from mismatched sequences. This biosensing strategy exhibits good reproducibility and precision, and has been successfully applied for detection of target DNA in complex sample matrices. In addition, the nonlinear HCR based SPR biosensing methodology is extended to the detection of adenosine triphosphate (ATP) by aptamer recognition. Thus, the versatile method might become a potential alternative tool for biomolecule detection in medical research and early clinical diagnosis.

A label-free strategy for measuring the affinity between monoclonal antibody and hapten using microdialysis sampling combined with chemiluminescent detection

It is of great importance to measure antibody affinity in the course of screening monoclonal antibody (McAb) for immunotherapy, immunoassay and immunological purification. Herein, by using terbutaline mouse McAb as a model, a novel label-free strategy based on on-line microdialysis (MD) sampling combined with flow injection chemiluminescent detection was designed for measuring antibody affinity to hapten in a homogeneous system. After this McAb incubated with its hapten, the unbound hapten was sampled on-line by the MD probe and injected into the chemiluminescent detection system for quantification. The obtained concentrations of the unbound hapten were treated with Scatchard analysis and Klotz analysis to calculate the affinity constant. The MD probe showed a recovery of 26.2% for terbutaline under the chosen conditions. The affinity constants obtained using Scatchard analysis and Klotz analysis were 4.9×106M−1 and 4.9×106M−1, respectively, showing negligible difference. The obtained affinity constants indicated that the investigated McAb was an antibody with medium affinity. The designed strategy provided a simple, rapid and low-cost approach for direct measurement of antibody affinity. Furthermore, it avoided the decrease of affinity, which was encountered frequently in the conventional approaches based on probe labeling of McAb and protein conjugation of hapten.

Label-free chemiluminescent strategy for highly selective and sensitive detection of adenosine triphosphate by cofactor-dependent enzymatic ligation-triggered polymerase chain reaction

In this work, a novel label-free chemiluminescent (CL) assay system was developed for highly sensitive and selective detection of adenosine triphosphate (ATP). The strategy relies on the powerful signal amplification capability of rolling circle amplification (RCA), the preferential binding ability of graphene oxide (GO) to single-stranded DNA (ssDNA), and instantaneous derivatization reaction between phenylglyoxal (PGO) and guanine nucleobases (G). In the presence of ATP, the T4 DNA ligase catalyzes the ligation reaction between the two ends of the padlock probe producing a closed circular DNA template that initiates the RCA reaction with phi29 DNA polymerase and dNTP. Therein, many complementary copies of the circular template can be generated. The formed long single-stranded DNA which contained an amount of guanine bases could be adsorbed on the surface of GO forming DNA-GO complexes. And the CL signal of DNA-GO complexes can be obtained via the instantaneous derivatization reaction between PGO and guanine bases. The CL signal increased linearly with the concentration of ATP from 0.1 to 2.5nM with a detection limit of 0.03nM. The system also showed high specificity to ATP against its analogues such as CTP, GTP, UTP, AMP, ADP, BSA and CaM. In addition, ATP has been determined in diluted serum indicating the applicability of this assay.

Rapid and Label-Free Strategy to Isolate Aptamers for Metal Ions.

Generating aptamers that bind to specific metal ions is challenging because existing aptamer discovery methods typically require chemical labels or modifications that can alter the structure and properties of the ions. In this work, we report an aptamer discovery method that enables us to generate high-quality structure-switching aptamers (SSAs) that undergo a conformational change upon binding a metal ion target, without the requirement of labels or chemical modifications. Our method is more efficient than conventional selection methods because it enables direct measurement of target binding via fluorescence-activated cell sorting (FACS), isolating only the desired aptamers with the highest affinity. Using this strategy, we obtained a highly specific DNA SSA with ∼30-fold higher affinity than the best aptamer for Hg(2+) in the literature. We also discovered DNA aptamers that bind to Cu(2+) with excellent affinity and specificity. Both aptamers were obtained within four rounds of screening, demonstrating the efficiency of our aptamer discovery method. Given the growing availability of FACS, we believe our method offers a general strategy for discovering high-quality aptamers for other ions and small-molecule targets in an efficient and reproducible manner.

Synthesis and sensing integration: A novel enzymatic reaction modulated Nanoclusters Beacon (NCB) “Illumination” strategy for label-free biosensing and logic gate operation

A novel fluorescent label-free “turn-on” NAD+ and adenosine triphosphate (ATP) biosensing strategy is proposed by fully exploiting ligation triggered Nanocluster Beacon (NCB). In the presence of the target, the split NCB was brought to intact, which brought the C-rich sequence and enhancer sequence in close proximity resulting in the lightening of dark DNA/AgNCs (“On” mode). Further application was presented for logic gate operation and aptasensor construction. The feasibility was investigated by Ultraviolet–visible spectroscopy (UV–vis), Fluorescence, lifetime and High Resolution Transmission Electron Microscopy (HRTEM) etc. The strategy displayed good performance in the detection of NAD+ and ATP, with the detection limit of 0.002nM and 0.001mM, the linear range of 10–1000nM and 0.003–0.01mM, respectively. Due to the DNA/AgNCs as fluorescence reporter, the completely label-free fluorescent strategy boasts the features of simplicity and low cost, and showing little reliance on the sensing environment. Meanwhile, the regulation by overhang G-rich sequence not relying on Förster energy transfer quenching manifests the high signal-to-background ratios (S/B ratios). This method not only provided a simple, economical and reliable fluorescent NAD+ assay but also explored a flexible G-rich sequence regulated NCB probe for the fluorescent biosensors. Furthermore, this sensing mode was expanded to the application of a logic gate design, which exhibited a high performance for not only versatile biosensors construction but also for molecular computing application.

Pulse Proteolysis and Precipitation for Target Identification.

In recent years, phenotypic screening has assumed a leading role in drug discovery efforts. However, development of new drugs from bioactive compounds obtained in screening campaigns requires identification of the cellular targets responsible for their biological activities. A new energetics-based method for target identification is presented: pulse proteolysis and precipitation for target identification (PePTID). In this method, proteins incubated with or without a ligand and submitted to a brief proteolytic pulse are directly analyzed and compared using a label-free semiquantitative mass spectrometry strategy, dispensing the SDS-PAGE readout and greatly improving the throughput. As a proof-of-concept, we applied the PePTID method to identify ATP-binding proteins in Mycobacterium smegmatis, a model system for Mycobacterium tuberculosis, the etiological agent of tuberculosis.

Label-free electrochemical genosensor based on mesoporous silica thin film.

A novel label-free electrochemical strategy for nucleic acid detection was developed by using gold electrodes coated with mesoporous silica thin films as sensing interface. The biosensing approach relies on the covalent attachment of a capture DNA probe on the surface of the silica nanopores and further hybridization with its complementary target oligonucleotide sequence, causing a diffusion hindering of an Fe(CN)6 (3-/4-) electrochemical probe through the nanochannels of the mesoporous film. This DNA-mesoporous silica thin film-modified electrodes allowed sensitive (91.7A/M) and rapid (45min) detection of low nanomolar levels of synthetic target DNA (25fmol) and were successfully employed to quantify the endogenous content of Escherichia coli 16S ribosomal RNA (rRNA) directly in raw bacterial lysate samples without isolation or purification steps. Moreover, the 1-month stability demonstrated by these biosensing devices enables their advanced preparation and storage, as desired for practical real-life applications. Graphical abstract Mesoporous silica thin films as scaffolds for the development of novel label-free electrochemical genosensors to perform selective, sensitive and rapid detection of target oligonucleotide sequences. Application towards E. coli determination.

New strategy to address DNA-methyl transferase activity in ovarian cancer cell cultures by monitoring the formation of 5-methylcytosine using HPLC-UV.

Methylation of mammalian genomic DNA is catalyzed by DNA methyltransferases (DNMTs). Aberrant expression and activity of these enzymes has been reported to play an important role in the initiation and progression of tumors and its response to chemotherapy. Therefore, there is a great interest in developing strategies to detect human DNMTs activity. We propose a simple, antibody-free, label-free and non-radioactive analytical strategy in which methyltransferase activity is measured trough the determination of the 5-methylcytosine (5mC) content in DNA by a chromatographic method (HPLC-UV) previously developed. For this aim, a correlation between the enzyme activity and the concentration of 5mC obtained by HPLC-UV is previously obtained under optimized conditions using both, un-methylated and hemi-methylated DNA substrates and the prokaryotic methyltransferase M.SssI as model enzyme. The evaluation of the methylation yield in un-methylated known sequences (a 623bp PCR-amplicon) turned to be quantitative (110%) in experiments conducted in-vitro. Methylation of hemi-methylated and low-methylated sequences could be also detected with the proposed approach. The application of the methodology to the determination of the DNMTs activity in nuclear extracts from human ovarian cancer cells has revealed the presence of matrix effects (also confirmed by standard additions) that hampered quantitative enzyme recovery. The obtained results showed the high importance of adequate sample clean-up steps.

A highly sensitive and facile graphene oxide-based nucleic acid probe: Label-free detection of telomerase activity in cancer patient's urine using AIEgens

Molecular beacon (MB)-based sensing platforms that consist of a fluorogen-quencher pair play an important role in medical and biological researches. However, the synthesis of both fluorogen and quencher in the nucleic acid probes will increase the burden of organic synthesis works and induce the difficulties for precisely controlling the relative distance between fluorogen and quencher, which may lead to false-positive and false-negative results. In this work, initially we report a single labeled MB (FAM-MB, with carboxyfluorescein as fluorogen and without quencher) thus simplifies MBs with the aid of graphene oxide (GO) to detect telomerase activity. To further simplify this structure, namely label-free strategy, we design a facile, sensitive and selective platform using a label-free beacon (AIE-MB, without fluorogen and quencher), based on aggregation-induced emission fluorogen (silole-R). Upon the addition of telomerase, AIE-MB induced comb-like DNA structure leads to high aggregation of silole-R and thus exhibits strong fluorescence emission. By exploitation of this, we can detect telomerase with superior sensitivity and demonstrate their applications in bladder cancer diagnosis. Compared to single-labeled FAM-MB based telomerase activity assay, the label-free AIE-MB induced method could perform the sensitive detection with high signal-to-background ratio.

An ultra-facile and label-free immunoassay strategy for detection of copper (II) utilizing chemiluminescence self-enhancement of Cu (II)-ethylenediaminetetraacetate chelate

The establishment of facile, rapid, sensitive and cost-effective protocols for the detection of heavy metals is of great significance for human health and environmental monitoring. Hereby, an ultra-facile and label-free immunoassay strategy was designed for detecting heavy metal ion by using Cu (II) as the model analyte. Cu (II) reacted previously with ethylenediaminetetraacetate (EDTA) was captured by immobilized monoclonal antibody for Cu (II)-EDTA chelate. Then Cu (II) was detected based on the self-enhancing effect of Cu (II)-EDTA chelate to luminol-H2O2 chemiluminescence reaction. The CL intensity is linear relative with Cu (II) concentration in a very wide range of 1.0–1000ng/mL, with a detection limit of 0.33ng/mL (S/N=3). Since the specificity of this proposed strategy relied on both the specificity of monoclonal antibody and the specificity of luminol-H2O2 system, it could avoid interference from most common ions. The proposed method was used successfully to detect Cu (II) in traditional Chinese medicine and environmental water samples with acceptable recovery values of 82–113%. This proof-of-principle work demonstrated the feasibility of the label-free immunoassay for heavy metal ions, and opened a new avenue for rapid screening and field assay for drug safety, environmental monitoring and clinical diagnosis.

Ultrasensitive Visual Detection of HIV DNA Biomarkers via a Multi-amplification Nanoplatform.

Methodologies to detect disease biomarkers at ultralow concentrations can potentially improve the standard of living. A facile and label-free multi-amplification strategy is proposed for the ultrasensitive visual detection of HIV DNA biomarkers in real physiological media. This multi-amplification strategy not only exhibits a signficantly low detection limit down to 4.8 pM but also provides a label-free, cost-effective and facile technique for visualizing a few molecules of nucleic acid analyte with the naked eye. Importantly, the biosensor is capable of discriminating single-based mismatch lower than 5.0 nM in human serum samples. Moreover, the visual sensing platform exhibits excellent specificity, acceptable reusability and a long-term stability. All these advantages could be attributed to the nanofibrous sensing platform that 1) has a high surface-area-to-volume provided by electrospun nanofibrous membrane, and 2) combines glucose oxidase (GOx) biocatalysis, DNAzyme-catalyzed colorimetric reaction and catalytic hairpin assembly (CHA) recycling amplification together. This multi-amplification nanoplatform promises label-free and visual single-based mismatch DNA monitoring with high sensitivity and specificity, suggesting wide applications that range from virus detection to genetic disease diagnosis.

Label-free electrochemical multi-sites recognition of G-rich DNA using multi-walled carbon nanotubes–supported molecularly imprinted polymer with guanine sites of DNA

A novel label-free electrochemical strategy was designed for multi-sites recognizing the guanine-rich DNA (G-rich DNA) with surface molecular imprinting polymer composite, in which the multi-walled carbon nanotubes (MWNCTs) were regarded as supporting material and the molecularly imprinted polymer (MIP) with guanine sites of DNA was applied as recognition element. Firstly, the vinyl groups were grafted onto the surface of carboxylic acid-functionalized MWNCTs (MWCNTs-COOH) through chemical modification. Then, the composite of MWCNTs-MIP was fabricated by the selective copolymerization of methacrylic acid, ethylene glycol dimethacrylate and guanine on the vinyl group-functionalized MWNTs surface. MWCNTs-MIP was characterized by different techniques, including Fourier transform infrared (FTIR) spectroscopy, scanning electronic microscopy (SEM), cyclic voltammogram (CV) and electrochemical impedance spectroscopy (EIS), showing that the MWCNTs-MIP composite was successfully prepared. Under the optimized conditions, the imprinting factor was nearly 5.68 according to the comparative slopes obtained on MWCNT-MIP and MWCNTs-non-imprinted polymer (MWCNTs-NIP), indicating that MWCNTs-MIP exhibited high selectivity for G-rich DNA. Moreover, the MWCNTs-MIP composite had a relatively wide linear range over G-rich DNA concentration of 0.05–1μM and 5–30μM with a detection limit of 7.52nM (S/N=3). Furthermore, the novel electrochemical strategy based on imprinted composite has very excellent performance in real samples of human serum and human urine.

PCR-free and label-free fluorescent detection of telomerase activity at single-cell level based on triple amplification

As a universal biomarker for cancer diagnostics and cancer therapeutics, telomerase has attracted extensive attention concerning its detection and discovery of its inhibitors. Herein, we developed a PCR-free and label-free fluorescent strategy for facile, reliable and highly sensitive assay of human telomerase activity from crude cancer cell extracts. A G-quadruplex-selective fluorescent dye, N-methyl mesoporphyrin IX (NMM), was utilized as signal probe. Two hairpin probes with hidden G-quadruplex strand in their stem were designed as assembly components of strand displacement reaction (SDR). In this strategy, one telomerase elongation product contains several hexamer repeats which can hybridize with numerous assistant DNA to release a lot of trigger DNA (T-DNA) of SDR for achieving first step amplification. Then, strand displacement reaction led to the formation of G-quadruplex at the both end of two hairpin DNA probes for realizing second step amplification. Finally, the re-released T-DNA initiated another cycle of SDR, resulting in a significant increase in the fluorescence intensity of NMM. By taking advantage of triple signal amplification, the telomerase activity in the HeLa extracts equivalent to 1-3000 cells was detected in homogeneous solution. Telomerase activities of different cell lines, including cancer cells and normal cell, were also successfully evaluated. Meanwhile, the inhibition effect of 3′-azido-3′-deoxythymidine (AZT) was also investigated. Therefore, it offers a simple and reliable method for detecting telomerase activity at single-cell level without complex pre-modification of probe and enzyme auxiliary signal amplification, which has the merits of simplicity, rapid response, low cost and high reliability.