Label-free Strategy Research Articles

Antibody-powered lighting-up fluorescence immunosensor based on hemin/G-quadruplex-quenched DNA-hosted dual silver nanoclusters as emitters

Immunosensors with many merits such as simplicity, high sensitivity and selectivity are intriguing for the detection of antibodies, especially bivalent antibody (e.g. anti-digoxin antibody, DigA). In this work, we report a sensitive immunosensor for DigA based on the switchable fluorescence of DNA-templated silver nanoclusters (AgNCs) modulated by hemin/G-quadruplex (hGq). To explore the fluorescing performance of one hGq-quenching dual-AgNCs (d-AgNCs), here we design a functional hosting DNA strand (HGH) encoding with one centered G-rich segment for forming hGq complex in the presence of hemin and K+, two terminated C-rich template sequences for the identical clustering of d-AgNCs, and two flanked symmetric domains for specific recognizing and linking. Because of the photoexcited electron transfer process to the stacked hGq, the originally presynthesized d-AgNCs colocalized in two ends are non-emissive, which are retainable after complementarily hybridizing with two recognizable single strands that are both tagged with a digoxigenin hapten, a Fab fragment. Upon introducing DigA, the steric strain associated with the affinity binding of two haptens results in the opening and stretch of hGq in a coil spacer, thereby lighting-up the d-AgNCs fluorescence. Based on one-step signal switch stimulated by targeting antibody, this label-free immunosensor strategy is of high specificity and sensitivity with low picomolar detection limit, as well as simplification, rapidness and cost-effectiveness.

Label-free SERS strategy for rapid detection of capsaicin for identification of waste oils

Identification of waste oils is challenging in the field of food safety due to the lack of common markers and straightforward analytical methods. Herein, we developed a novel label-free surface-enhanced Raman spectroscopy (SERS) strategy to identify waste oils using Ag nanoparticles solution (Ag NPs sol.) as a SERS substrate to significantly enhance the Raman signal of capsaicin marker molecule usually contained in the waste oils. The enhanced signal was directly detected by a portable Raman spectrometer with the limit of detection (LOD) of 2.9 μg L−1 within 10 min. Concentration-dependent SERS investigation showed the linear relationship between the SERS signal intensity of the characteristic peaks and the concentrations of capsaicin in the range of 10–2500 μg L−1 and the correlation coefficient was 0.9895. Our findings show the sensitivity, accessibility, and reliability of this method for the rapid identification of waste oils and furthermore for the practical applications in the field of food safety.

A Label-Free Fluorescent AND Logic Gate Aptasensor for Carbohydrate Antigen 15-3 Detection Based on Graphene Oxide.

Molecular logic gate always makes use of fluorescent dyes to realize fluorescence signals. The labeling of the fluorophore is relatively expensive, resulting in low yield, and singly labeled impurities affect the affinity between the target and the aptamer. Label-free fluorescent aptamer biosensor strategy has attracted widespread interest due to lower cost and simplicity. Herein, we have designed an AND logic gate fluorescent aptasensor for detecting carbohydrate antigen 15-3(CA15-3) based on label-free fluorescence signal output. A hairpin DNA probe consists of CA15-3 aptamer and partly anti-CA15- 3 aptamer sequences as a long stem and G-rich sequences of the middle ring as a quadruplexforming oligomer. G-rich sequences can fold into a quadruplex by K+, and then G-quadruplex interacts specifically with N-methylmesoporphyrin IX(NMM), leading to a dramatic increase in fluorescence of NMM. With CA15-3 and NMM as the two inputs, the fluorescence intensity of the NMM is the output signal. Lacking CA15-3 or NMM, there is no significant fluorescence enhancement, and the output of the signal is "0". The fluorescence signal dramatically increases and the output of the signal is "1" only when CA15-3 protein and NMM are added at the same time. This biosensor strategy was observed to possess selectivity and high sensitivity for detecting CA15-3 protein from 10 to 500 U mL-1 and the detection limit was found to be 10 U mL-1, which also showed good reproducibility in spiked human serum. In summary, the proposed AND logic gate fluorescent aptasensor could specifically detect CA15-3.

Single-atom sites on perovskite chips for record-high sensitivity and quantification in SERS

Surface enhanced Raman scattering (SERS) is a rapid and nondestructive technique that is capable of detecting and identifying chemical or biological compounds. Sensitive SERS quantification is vital for practical applications, particularly for portable detection of biomolecules such as amino acids and nucleotides. However, few approaches can achieve sensitive and quantitative Raman detection of these most fundamental components in biology. Herein, a noble-metal-free single-atom site on a chip strategy was applied to modify single tungsten atom oxide on a lead halide perovskite, which provides sensitive SERS quantification for various analytes, including rhodamine, tyrosine and cytosine. The single-atom site on a chip can enable quantitative linear SERS responses of rhodamine (10−6−1 mmol L−1), tyrosine (0.06–1 mmol L−1) and cytosine (0.2–45 mmol L−1), respectively, which all achieve record-high enhancement factors among plasmonic-free semiconductors. The experimental test and theoretical simulation both reveal that the enhanced mechanism can be ascribed to the controllable single-atom site, which can not only trap photoinduced electrons from the perovskite substrate but also enhance the highly efficient and quantitative charge transfer to analytes. Furthermore, the label-free strategy of single-atom sites on a chip can be applied in a portable Raman platform to obtain a sensitivity similar to that on a benchtop instrument, which can be readily extended to various biomolecules for low-cost, widely demanded and more precise point-of-care testing or in-vitro detection.Electronic Supplementary MaterialSupplementary material is available for this article at 10.1007/s40843-022-1968-5 and is accessible for authorized users.

Rationally Driven Drug Nonradiative Decay via a Label-Free Polyprodrug Strategy to Renew Tumor Cascade Photothermal-Chemotherapy.

Drugs are frequently used for only chemotherapy that ignores their photophysical properties that potentially endow them with other therapeutic potency. Additionally, current photothermal-chemotherapy replies on the codelivery of drugs and photothermal agents, but their spatiotemporal delivery and precise release is unsatisfactory. Herein, label-free doxorubicin (DOX) polyprodrug nanoparticles (DPNs) are formulated from disulfide bonds-tethered DOX polyprodrug amphiphiles (PDMA-b-PDOXM). Benefiting from boosted nonradiative decay of high-density DOX, significant fluorescence quenching and photothermal effects are observed for DPNs without common photothermal agents. Upon cellular uptake and laser irradiation, the heat can promote lysosomal escape of DPNs into reductive cytosol, whereupon free DOX is released to activate chemotherapy and fluorescence, achieving rational cascade photothermal-chemotherapy. The current label-free polyprodrug strategy can make full use of drugs; it provides an alternative insight to extend the therapeutic domain of drugs.

Morphologically Flex Sm-MOF Based Electrochemical Immunosensor for Ultrasensitive Detection of a Colon Cancer Biomarker.

Despite having the potential to synthesize stable metal-organic frameworks (MOFs), rare earth metal-based MOFs have not been exploited extensively. Owing to the high coordination numbers, the MOFs can generate a suitable coordination environment for various applications. Herein, samarium (Sm)-based MOFs were synthesized with three different organic linkers, namely, trimesic acid (TMA), meso-tetra(4-carboxyphenyl)porphine (TCPP), and 1,3,6,8-tetra(4-carboxylphenyl) pyrene(TBPy) by the solvothermal approach. The morphologies of Sm-TMA MOF, Sm-TCPP MOF, Sm-TBPy MOF were rod-shaped, cubic consisting of stacked 2D layers, and spherical made of small cubic structures, respectively. After the electrochemical properties of the synthesized MOFs were investigated, the MOFs were used to fabricate immunosensors for detection of carcinoembryonic antigen using a label-free signaling strategy. The immunosensors exhibited a wide linear detection range and a lower detection limit. The exhibited reproducibility and selectivity of the immunosensors were within the tolerable limits. The established label-free immunosensor has been successfully applied for detection of carcinoembryonic antigen in human serum samples, demonstrating that the rare earth metal-based MOFs are promising for construction of biosensors for medical diagnosis.

A label-free and modification-free ratiometric electrochemical strategy for enhanced natural enzyme detection using a bare electrode and nanozymes system.

Ratiometric electrochemical assays have been demonstrated to be more sensitive and selective in various sensing events, mainly due to their affordable built-in correction and good self-reference capability. But it is known that complicated modification and labeling operations usually are necessary for the construction of ratiometric electrochemical assays, therefore is a hot and important issue needing consideration carefully. We herein report a new yet simple bare electrode-based ratiometric electrochemical bioassay to achieve sensitive and selective analysis of alkaline phosphatase (ALP), using a liquid phase system that contains CoOOH nanozymes and commercially available indicator substrate. This proposed bioassay works based on the ratiometric change of dual electrochemical signals, arising from an exclusive target ALP-triggered hydrolysis of electrochemical substrate p-nitrophenyl phosphate (PNPP). In this design, the two hydrolyzed products of electrochemically active p-nitrophenol (PNP) and electrochemically inactive phosphate anion (PO43-) are responsible together for the ratiometric electrochemical analysis of ALP. PNP exhibits a straightforward current response toward ALP content; however, PO43- cannot show a direct electrochemical signal thus is rationally designed to offer an alternative response by linking it with the specific CoOOH nanozyme-catalyzed reaction of 3,3',5,5'-tetramethylbenzidine (TMB) and H2O2, in which the nanozyme-catalyzed product oxTMB shows a direct reduction current at the GCE, and significantly decreases with increasing PO43- species due to the good inhibition of PO43- toward CoOOH nanozyme activity. As a result, a ratiometric electrochemical strategy for ALP analysis with a low limit of detection of 0.366 U/L (S/N = 3) was successfully achieved by integrating the above direct and indirect dual electrochemical responses. This developed bioassay can allow the quantitative diagnosis of ALP activity especially with a label-free and modification-free merit, therefore paving the way for simple, convenient, and portable electroanalytical tools in biosensing design and application.

Innovative FO-SPR Label-free Strategy for Detecting Anti-RBD Antibodies in COVID-19 Patient Serum and Whole Blood.

The ongoing COVID-19 pandemic has emphasized the urgent need for rapid, accurate, and large-scale diagnostic tools. Next to this, the significance of serological tests (i.e., detection of SARS-CoV-2 antibodies) also became apparent for studying patients’ immune status and past viral infection. In this work, we present a novel approach for not only measuring antibody levels but also profiling of binding kinetics of the complete polyclonal antibody response against the receptor binding domain (RBD) of SARS-CoV-2 spike protein, an aspect not possible to achieve with traditional serological tests. This fiber optic surface plasmon resonance (FO-SPR)-based label-free method was successfully accomplished in COVID-19 patient serum and, for the first time, directly in undiluted whole blood, omitting the need for any sample preparation. Notably, this bioassay (1) was on par with FO-SPR sandwich bioassays (traditionally regarded as more sensitive) in distinguishing COVID-19 from control samples, irrespective of the type of sample matrix, and (2) had a significantly shorter time-to-result of only 30 min compared to >1 or 4 h for the FO-SPR sandwich bioassay and the conventional ELISA, respectively. Finally, the label-free approach revealed that no direct correlation was present between antibody levels and their kinetic profiling in different COVID-19 patients, as another evidence to support previous hypothesis that antibody-binding kinetics against the antigen in patient blood might play a role in the COVID-19 severity. Taking all this into account, the presented work positions the FO-SPR technology at the forefront of other COVID-19 serological tests, with a huge potential toward other applications in need for quantification and kinetic profiling of antibodies.

Capacitive Aptasensor Coupled with Microfluidic Enrichment for Real-Time Detection of Trace SARS-CoV-2 Nucleocapsid Protein.

The pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has lasted for almost 2 years. Stemming its spread has posed severe challenges for clinical virus detection. A long turnaround time, complicated operation, and low accuracy have become bottlenecks in developing detection techniques. Adopting a direct antigen detection strategy, we developed a fast-responding and quantitative capacitive aptasensor for ultratrace nucleocapsid protein detection based on a low-cost microelectrode array (MEA) chip. Employing the solid–liquid interface capacitance with a sensitivity of picofarad level, the tiny change on the MEA surface can be definitively detected. As a result, the limit of detection reaches an ultralow level of femtogram per milliliter in different matrices. Integrated with efficient microfluidic enrichment, the response time of this sensor from the sample to the result is shortened to 15 s, completely meeting the real-time detection demand. Moreover, the wide linear range of the sensor is from 10–5 to 10–2 ng/mL, and a high selectivity of 6369:1 is achieved. After application and evaluation in different environmental and body fluid matrices, this sensor and the detection method have proved to be a label-free, real-time, easy-to-operate, and specific strategy for SARS-CoV-2 screening and diagnosis.

Synthesis of a metal oxide affinity chromatography magnetic mesoporous nanomaterial and development of a one-step selective phosphopeptide enrichment strategy for analysis of phosphorylated proteins

Protein phosphorylation is one of the most vital protein post-translational modifications, which regulates numerous biological processes including protein structure transformation, molecular interaction, cell proliferation and apoptosis, as well as various malignant diseases. Though mass spectrometry (MS)-based technologies provide a feasible strategy to analyze phosphorylated proteins, label-free quantitative analysis for phosphopeptides in biological samples is still a challenging task due to sample losses from complicated preparation and necessary phosphopeptide enrichment. In this study, magnetic mesoporous TiO2 microspheres, Fe3O4@nSiO2@mSiO2@TiO2, a metal oxide affinity chromatography material, were synthesized with uniform particle size of 500 nm. The synthesized material exhibited good selectivity (molar ratio of β-casein:BSA = 1:2000, great sensitivity (0.5 fmol μL−1β-casein), and good adsorption capacity (120 mg g−1). Moreover, the proper mesopores on the surface of the enrichment material displayed the unique property of size-exclusion, which could ‘capture’ the trypsin and form a temporary immobilized enzyme reactor, reducing the tryptic digestion procedure from at least 16 h to 2 h. Based on the synthesized material, a facile one-step selective phosphopeptide enrichment method was successfully developed and consisted with protein digestion and phosphopeptide enrichment, which was further applied to selectively enrich phosphopeptides from the complex biological samples of nonfat milk and HepG2 cells, indicating its great feasibility and potential of applications in real sample analysis. More importantly, combining with MS, a label-free phosphoproteomic quantitative strategy was established and performed for determination of the mixture samples containing BSA and various concentrations of β-casein. The results showed that the obtained calibration curve was linear with R = 0.9987 by plotting the intensities of unique phosphopeptide of β-casein (m/z 2061.77) versus the concentrations of β-casein. Our study will provide a novel analytical strategy for selective enrichment of phosphopeptides and facile label-free relative quantitative analysis of phosphorylated proteins.

Label-free fluorescent aptasensor for chloramphenicol based on hybridization chain reaction amplification and G-quadruplex/N-methyl mesoporphyrin IX complexation.

The use of the broad-spectrum antibiotic chloramphenicol (CAP) in food is strictly regulated or banned in many countries. Herein, for the sensitive, rapid, and specific detection of CAP in milk, a label-free fluorescence strategy was established based on guanine (G)-quadruplex/N-methyl mesoporphyrin IX (NMM) complex formation and hybridization chain reaction (HCR) amplification. In this system, CAP can specifically bind to an aptamer (Apt) to release an Apt-C sequence from double-stranded DNA (Apt·Apt-C). Apt-C, can further hybridize with a functional hairpin DNA probe to release a primer sequence. The released primer sequence causes HCR and the formation of a nicked double-helix polymer, which contains G-quadruplex DNA. The recognition of G-quadruplex DNA by the NMM fluorochrome results in fluorescence enhancement. Consequently, CAP can be quantitatively detected by measuring the fluorescence intensity at 612 nm. The reliability of the aptasensor method was confirmed by comparison with an enzyme-linked immunosorbent assay. The proposed aptasensor was found to have a limit of detection of 0.8 pg mL−1 for CAP. Moreover, when the aptasensor was applied to the detection of CAP in milk samples, the average recoveries were 99.8–108.3% with relative standard deviations of 4.5–5.2%. Thus, this CAP detection method, which is rapid with high sensitivity and selectivity, has considerable potential for a wide range of food analysis applications.

Peptidomics Analysis Reveals Serum Biomarkers in Spinal Cord Injury Patients.

Spinal cord injury (SCI) is a severe traumatic disease, always resulting in neuronal injury. In this study, we aimed to exhibit a peptidome profile of serum from patients with SCI. A label-free peptidomics strategy was used to analyze the differentially expressed peptides (DEPs). Then, gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) enrichment analyses was used to evaluate the function of the peptides precursors proteins. Also, the protein-protein interaction networks were mapped using STRING database. Finally, parallel reaction monitoring assays were used to validate the expression of candidate peptides. We identified 217 DEPs including 29 upregulated peptides and 188 downregulated peptides in SCI group. Many pathways such as Platelet activation, Complement and coagulation cascades, Focal adhesion were enriched. Seven peptides including PSPRPSP, RPPGFSP, DKPDMAEIEKFDKSKLK, STTAVVTNPKE, GHAGAQGPPGPPG, SMPPAQQQITS and SKVLPIQDNVSK were significantly changed between SCI patients and healthy people. Peptidomics provide a powerful tool to find the variation of SCI. RPPGFSP, DKPDMAEIEKFDKSKLK and SMPPAQQQITS may play important roles in SCI. However, the specific function of these peptides and whether they can be used as therapeutic targets for SCI need to be further investigated.

Raman spectroscopy reveals phenotype switches in breast cancer metastasis.

The accurate analytical characterization of metastatic phenotype at primary tumor diagnosis and its evolution with time are critical for controlling metastatic progression of cancer. Here, we report a label-free optical strategy using Raman spectroscopy and machine learning to identify distinct metastatic phenotypes observed in tumors formed by isogenic murine breast cancer cell lines of progressively increasing metastatic propensities.Methods: We employed the 4T1 isogenic panel of murine breast cancer cells to grow tumors of varying metastatic potential and acquired label-free spectra using a fiber probe-based portable Raman spectroscopy system. We used MCR-ALS and random forests classifiers to identify putative spectral markers and predict metastatic phenotype of tumors based on their optical spectra. We also used tumors derived from 4T1 cells silenced for the expression of TWIST, FOXC2 and CXCR3 genes to assess their metastatic phenotype based on their Raman spectra.Results: The MCR-ALS spectral decomposition showed consistent differences in the contribution of components that resembled collagen and lipids between the non-metastatic 67NR tumors and the metastatic tumors formed by FARN, 4T07, and 4T1 cells. Our Raman spectra-based random forest analysis provided evidence that machine learning models built on spectral data can allow the accurate identification of metastatic phenotype of independent test tumors. By silencing genes critical for metastasis in highly metastatic cell lines, we showed that the random forest classifiers provided predictions consistent with the observed phenotypic switch of the resultant tumors towards lower metastatic potential. Furthermore, the spectral assessment of lipid and collagen content of these tumors was consistent with the observed phenotypic switch.Conclusion: Overall, our findings indicate that Raman spectroscopy may offer a novel strategy to evaluate metastatic risk during primary tumor biopsies in clinical patients.

Target-induced activation of DNAzyme for highly sensitive colorimetric detection of bleomycin via DNA scission.

In this work, a label-free and sensitive colorimetric sensing strategy for the detection of bleomycin (BLM) was developed on the basis of BLM-mediated activation of G-quadruplex DNAzyme via DNA strand scission. A G-quadruplex based hairpin probe (G4HP) containing the scission site (5′-GT-3′) of BLM at the loop region and guanine (G)-rich sequences at its 5′-end was employed in this protocol. In the presence of BLM, it may cleave the 5′-GT-3′ site of the hairpin probe with Fe(ii) as a cofactor, releasing the G-tetrads DNA fragment, which may further bind hemin to form a catalytic G-quadruplex-hemin DNAzyme. The resultant G-quadruplex DNAzyme has notable peroxidase-like activity, which effectively catalyzes the oxidation of 2,2′-azino-bis(3-ethylbenzothiozoline-6-sulfonic acid) (ABTS) by H2O2 to produce the blue-green-colored free-radical cation (ABTS·+). Therefore, the detection of BLM can be achieved by observing the color transition with the naked eye or measuring the absorbance at a wavelength of 420 nm using a UV-Vis spectrophotometer. Attributing to the specific BLM-induced DNA strand scission and the effective locking of G-tetrads in the stem of the G4HP, the colorimetric sensing strategy exhibits high sensitivity and selectivity for detection of BLM in human serum samples, which might hold great promise for BLM assay in biomedical and clinical research.

A label-free strategy for visual genotyping based on phosphate induced coloration reaction.

Genetic variation plays a crucial role in disease occurrence and development. However, current genotyping strategies not only require a long turnaround time for DNA purification, but also depend on sophisticated apparatus and complex data interpretation, which seriously limits their application in point of care diagnostic test scenarios. In this study, by integrating phosphate induced coloration reaction and loop-mediated isothermal amplification, a rapid and portable strategy for straightforward genotyping has been established to cater to the demand of precision medicine. By employing phosphate ions produced during the amplification as a signal generator, not only can the genotyping result be interpreted with only naked eye from a low-cost label-free strip, but also the amplification efficiency is increased to facilitate genotyping with a robust biological specimen ignoring DNA polymerase inhibitors. Moreover, the introduction of alkaline lysis for DNA release allows whole blood to be identified accurately avoiding DNA purification. As a proof of concept, the insertion/deletion polymorphisms of the angiotensin-converting enzyme, a crucial factor associated with cardiovascular and cerebrovascular diseases, has been selected as a model to evaluate the performance of this method. Accurate results can be obtained from as low as 1 ng genomic DNA within 30 min. For clinical specimen detection, a concordance rate up to 100% has been found compared with PCR-based electrophoresis. Thus, this novel strategy may serve as a promising tool for straightforward genotyping to provide timely diagnostic information, especially in resource-poor medical institutions.

A rapid and label-free DNA-based interference reduction nucleic acid amplification strategy for viral RNA detection

Common reference methods for COVID-19 diagnosis include thermal cycling amplification (e.g. RT-PCR) and isothermal amplification methods (e.g. LAMP and RPA). However, they may not be suitable for direct detection in environmental and biological samples due to background signal interference. Here, we report a rapid and label-free interference reduction nucleic acid amplification strategy (IR-NAAS) that exploits the advantages of luminescent iridium(III) probes, time-resolved emission spectroscopy (TRES) and multi-branch rolling circle amplification (mbRCA). Using IR-NAAS, we established a luminescence approach for diagnosing COVID-19 RNAs sequences RdRp, ORF1ab and N with a linear range of 0.06–6.0 × 105 copies/mL and a detection limit of down to 7.3 × 104 copies/mL. Moreover, the developed method was successfully applied to detect COVID-19 RNA sequences from various environmental and biological samples, such as domestic sewage, and mice urine, blood, feces, lung tissue, throat and nasal secretions. Apart from COVID-19 diagnosis, IR-NAAS was also demonstrated for detecting other RNA viruses, such as H1N1 and CVA10, indicating that this approach has great potential approach for routine preliminary viral detection.

60-nt DNA Direct Detection without Pretreatment by Surface-Enhanced Raman Scattering with Polycationic Modified Ag Microcrystal Derived from AgCl Cube.

Direct detection of long-strand DNA by surface-enhanced Raman scattering (SERS) is a valuable method for diagnosis of hereditary diseases, but it is currently limited to less than 25-nt DNA strand in pure water, which makes this approach unsuitable for many real-life applications. Here, we report a 60-nt DNA label-free detection strategy without pretreatment by SERS with polyquaternium-modified Ag microcrystals derived from an AgCl cube. Through the reduction-induced decomposition, the size of the about 3 × 3 × 3 μm3 AgCl cube is reduced to Ag, and the surface is distributed with the uniform size of 63 nm silver nanoparticles, providing a large area of a robust and highly electromagnetic enhancement region. The modified polycationic molecule enhances the non-specific electrostatic interaction with the phosphate group, thereby anchoring DNA strands firmly to the SERS enhanced region intactly. As a result, the single-base recognition ability of this strategy reaches 60-nt and is successfully applied to detect thalassemia-related mutation genes.

Nanopipette-Based Nanosensor for Label-Free Electrochemical Monitoring of Cell Membrane Rupture under H2O2 Treatment.

H2O2 is an essential signaling molecule in living cells that can cause direct damage to lipids, proteins, and DNA, resulting in cell membrane rupture. However, current studies mostly focus on probe-based sensing of intracellular H2O2, and these methods usually require sophisticated probe synthesis and instruments. In particular, local H2O2 treatment induces cell membrane rupture, but the level of cell membrane destruction is unknown because the mechanical properties of the cell membrane are difficult to accurately determine. Therefore, highly sensitive and label-free methods are required to measure and reflect mechanical changes in the cell membrane. Here, using an ultrasmall quartz nanopipette with a tip diameter less than 90 nm as a nanosensor, label-free and noninvasive electrochemical single-cell measurement is achieved for real-time monitoring of cell membrane rupture under H2O2 treatment. By spatially controlling the nanopipette tip to precisely approach a specific location on the membrane of a single living cell, stable cyclic membrane oscillations are observed under a constant direct current voltage. Specifically, upon nanopipette advancement, the mechanical status of the cell membrane can be sensibly displayed by continuous current versus time traces. The electrical signals are collected and processed, ultimately revealing the mechanical properties of the cell membrane and the degree of cell apoptosis. This nanopipette-based nanosensor paves the way for developing a facile, label-free, and noninvasive strategy to assay the mechanical properties of the cell membrane during external stimulation at the single-cell level.

In situ label-free and sensitive detection assay for cell apoptosis via polyadenosine-coralyne fluorescence enhancement strategy

Cell apoptosis detection is vital for biological analysis and clinical application; some detection assays are already commercially available. However, it is still far from perfect and needs further improvement for less cost, time-consuming and operation demanding. TUNEL, a high market share cell apoptosis assay, depends on adulteration fluorescent labelling dUTP by terminal deoxynucleotidyl transferase(TdT) which randomly adds deoxyribonucleoside triphosphates (dNTPs) at the 3′-OH terminal of ssDNA with a template-free manner. Based on our previous work, we adopted a label-free strategy to reduce the cost and operation maintenance of TUNEL and developed a facile, rapid, convenient and in-situ assay for cell apoptosis.

Identification of novel ADAMTS1, ADAMTS4 and ADAMTS5 cleavage sites in versican using a label-free quantitative proteomics approach

The chondroitin sulfate proteoglycan versican is important for embryonic development and several human disorders. The versican V1 splice isoform is widely expressed and cleaved by ADAMTS proteases at a well-characterized site, Glu441-Ala442. Since ADAMTS proteases cleave the homologous proteoglycan aggrecan at multiple sites, we hypothesized that additional cleavage sites existed within versican. We report a quantitative label-free approach that ranks abundance of liquid chromatography-tandem mass spectrometry (LC-MS/MS)-identified semi-tryptic peptides after versican digestion by ADAMTS1, ADAMTS4 and ADAMTS5 to identify site-specific cleavages. Recombinant purified versican V1 constructs were digested with the recombinant full-length proteases, using catalytically inactive mutant proteases in control digests. Semi-tryptic peptide abundance ratios determined by LC-MS/MS in ADAMTS:control digests were compared to the mean of all identified peptides to obtain a z-score by which outlier peptides were ranked, using semi-tryptic peptides identifying Glu441 -Ala442 cleavage as the benchmark. Tryptic peptides with higher abundance in control digests supported cleavage site identification. We identified several novel cleavage sites supporting the ADAMTS1/4/5 cleavage site preference for a P1-Glu residue in proteoglycan substrates. Digestion of proteins in vitro and application of this z-score approach is potentially widely applicable for mapping protease cleavage sites using label-free proteomics. SignificanceVersican abundance and turnover are relevant to the pathogenesis of several human disorders. Versican is cleaved by A Disintegrin-like And Metalloprotease with Thrombospondin type 1 motifs (ADAMTS) family members at Glu441-Ala442, generating a bioactive proteoform called versikine, but additional cleavage sites and the site-specificity of individual ADAMTS proteases is unexplored. Here, we used a label-free proteomics strategy to identify versican cleavage sites for 3 ADAMTS proteases, applying a novel z-score-based statistical approach to compare the protease digests of versican to controls (digests with inactive protease) using the known protease cleavage site as a benchmark. We identified 21 novel cleavage sites that had a comparable z-score to the benchmark. Given the functional significance of versikine, they represent potentially significant cleavages and helped to refine a substrate site preference for each protease.The z-score approach is potentially widely applicable for discovery of site-specific cleavages within an purified protein or small ensemble of proteins using any protease.