Real-time observation of integrin bending/unbending conformational changes on living cells

Introduction Integrins are a large family of adhesion molecules broadly expressed on the surface of a wide variety of cells as heterodimers. Binding of integrins to ligands provides not only mechanical anchorage for the cell to another cell or

The Electrochemical Impedance Behavior of the Living Cells Escherichia Coli

The semiconductive characteristics of clectron-transfrring proteins in living cells E coli was investigated by electrochemsical impedance spectroscopy(EIS). We found that the electrochemical impedance of living cells as a function of temprature followed the Arrhenius equation for semiconductors. This result shows a strong evidence to prove the semiconductive behavior of

Iron-Imprinted Single-Atomic Site Catalyst-Based Nanoprobe for Detection of Hydrogen Peroxide in Living Cells

Fe-based single-atomic site catalysts(SASCs),with the natural metalloproteases-like active site structure,have attracted widespread attention in biocatalysis and biosensing.Precisely,controlling the isolated single-atom Fe-N-C active site structure is crucial to improve the SASCs’performance.In this work,we use a facile ion-imprinting method(IIM)to synthesize isolated Fe-N-C single-atomic site catalysts(IIM-Fe-SASC).With this method,the ion-imprinting process can precisely control ion at the atomic level and form numerous well-defined single-atomic Fe-N-C sites.The IIM-Fe-SASC shows better peroxidase-like activities than that of non-imprinted references.Due to its excellent properties,IIM-Fe-SASC is an ideal nanoprobe used in the colorimetric biosensing of hydrogen peroxide(H_(2)O_(2)).Using IIM-Fe-SASC as the nanoprobe,in situ detection of H_(2)O_(2)generated from MDA-MB-231 cells has been successfully demonstrated with satisfactory sensitivity and specificity.This work opens a novel and easy route in designing advanced SASC and provides a sensitive tool for intracellular H_(2)O_(2)detection.

DNA tetrahedron-based split aptamer probes for reliable imaging of ATP in living cells

Accurate detection and imaging of adenosine triphosphate(ATP)expression levels in living cells is of great value for understanding cell metabolism,physiological activities,and pathologic mechanisms.Here,we developed a DNA tetrahedron-based split aptamer probe(TD probe)for ratiometric fluorescence imaging of ATP in living cells.The TD probe is constructed by hybridizing two split ATP aptamer probes(Apt-a and Apt-b)to a DNA tetrahedron assembled by four DNA oligonucleotides(T1,T2,T3 and T4).In the presence of ATP,the TD probe will alter its structure from the open to closed state,thus bringing the separated donor and acceptor fluorophores into close proximity for high fluorescence resonance energy transfer(FRET)signals.The TD probe exhibits low cytotoxicity,efficient cell internalization and good biological stability.Moreover,based on the FRET“off”to“on”signal output mode,the TD probe can effectively avoid false-positive signals from complex biological matrices,which is significant for long-term reliable imaging in living cells.In addition,by changing the split aptamers attached to DNA tetrahedron,the proposed strategy may be extended for detecting various intracellular targets.Collectively,this strategy provides a valuable sensing platform for biomarkers analysis in living cells,thus having great potential for early clinical diagnosis and therapeutic evaluation.

A highly sensitive DNA-AIEgen-based ＂turn-on＂ fluorescence chemosensor for amplification analysis of Hg^2+ ions in real samples and living cells

Functional nucleic acids(FNAs)-based biosensors have shown great potential in heavy metal ions detection due to their low-cost and easy to operate merits. However, in most FNAs based fluorescence probes, the ingenious designs of double-labeled(fluorophore and quencher group) DNA sequence, not only bring the annoyance of organic synthesis, but also restrict its use as a robust biosensor in practical duties. In this paper, we design a simple AIEgens functional nucleic acids(AFNAs) probe which consists of only fluorogen but no quencher group. With the help of duplex-specific nuclease(DSN) enzyme based target recycling, high fluorescence signal and superior sensitivity towards Hg^(2+) are achieved. This robust assay allows for sensitive and selective detection of Hg^(2+) in real water samples and mapping of intracellular Hg^(2+), without double-labeling of oligonucleotide with a dye-quencher pair, nor the multiple assay steps.

A unique two-photon fluorescent probe based on ICT mechanism for imaging palladium in living cells and mice

Palladium(0)as one of the vital transition metals,is employed in numerous industries,such as drug synthesis,aerospace high-tech field and automobile industry.When the Pd(0)enter into the body,it will bind with thiol-containing amino acids,DNA,RNA,and other biomolecules damaging to human health.Thus,developing a novel tool for monitoring and imaging of Pd(0)in vivo is very urgent.In the work,based on a intramolecular charge transfer(ICT)mechanism a two-photon fluorescent probe NIPd had been designed and synthesized for the recognition Pd(0).In vitro experiments data displayed that probe NIPd exhibited a 13-fold fluorescent increase for Pd(0)in 30 min in the aqueous solution with a detection limit of 16 nmol/L.It also showed the outstanding selectivity and antijamming performance.More importantly,NIPd could be served as a two-photon fluorescent probe for real-time monitoring Pd(0)in living cells and mice.

Two-dimensional coordination polymer-based nanosensor for sensitive and reliable nucleic acids detection in living cells

A reliable and sensitive strategy which can assess nucleic acid levels in living cells would be essential for fundamental research of biomedical applications. Some nanomaterial-based fluorescence biosensors recently developed for detecting nucleic acids, however, are often with expensive, complicated and timeconsuming preparation process. Here, by using a facile bottom-up synthesis method, a two-dimensional(2 D) coordination polymer(CP) nanosheet, [Cu(tz)](Htz = 1,2,4-triazole), was successfully prepared after optimizing reaction conditions. These ultrathin CP nanosheets with thickness of 4.7 ± 1.1 nm could readily form nanosensors by assembly with DNA probes, which exhibited a low limit of detection(LOD)for p53 DNA fragment as 144 pmol/L. Furthermore, by integrating [Cu(tz)] nanosheets with hybridization chain reaction(HCR) probes, mi R-21, one kind of micro RNA upregulated in many cancer cells, can be sensitively detected with a LOD of 100 pmol/L and monitored in living cells, giving consistent results with those obtained by quantitative reverse-transcription polymerase chain reaction(q RT-PCR) analysis.Thus [Cu(tz)] nanosheets, which not only possess much better nucleic acids sensing performance than bulk cystals, but also exhibit nucleic acid delivery functions, could be used as a novel nanoplatform in biomedical imaging and sensing applications.

Non-contact mechanical and chemical analysis of single living cells by microspectroscopic techniques

Innovative label-free microspectroscopy,which can simultaneously collect Brillouin and Raman signals,is used to characterize the viscoelastic properties and chemical composition of living cells with sub-micrometric resolution.The unprecedented statistical accuracy of the data combined with the high-frequency resolution and the high contrast of the recently built experimental setup permits the study of single living cells immersed in their buffer solution by contactless measurements.The Brillouin signal is deconvoluted in the buffer and the cell components,thereby revealing the mechanical heterogeneity inside the cell.In particular,a 20%increase is observed in the elastic modulus passing from the plasmatic membrane to the nucleus as distinguished by comparison with the Raman spectroscopic marker.Brillouin line shape analysis is even more relevant for the comparison of cells under physiological and pathological conditions.Following oncogene expression,cells show an overall reduction in the elastic modulus(15%)and apparent viscosity(50%).In a proof-of-principle experiment,the ability of this spectroscopic technique to characterize subcellular compartments and distinguish cell status was successfully tested.The results strongly support the future application of this technique for fundamental issues in the biomedical field.

Research on Electric Impedance Spectroscopy of Living Cell Suspensions by a Chip with Microelectrodes

A microfabricated electrical impedance spectroscopy (EIS) chip with microelectrodes was developed.The substrate and the electrodes of the chip were made of glass and gold,respectively.The experimental results demonstrated that the EIS-chip could distinguish different solutions (physiological saline,culture medium,living cell suspension etc.) by scanning from 10Hz to 45kHz.A 6-element circuit model was used for fitting the real part and the imaginary part admittance curves of the living cell suspension.An actual circuit was also built and tested to verify the 6-element circuit model proposed.The micro-EIS chip has several advantages including the use of small sample volumes,high resolution and ease of operation.It shows good application prospects in the areas of cellular electrophysioiogy,drug screening and bio-sensors etc.

The emerging technology of biohybrid micro-robots:a review

In the past few decades,robotics research has witnessed an increasingly high interest in miniaturized,intelligent,and integrated robots.The imperative component of a robot is the actuator that determines its performance.Although traditional rigid drives such as motors and gas engines have shown great prevalence in most macroscale circumstances,the reduction of these drives to the millimeter or even lower scale results in a significant increase in manufacturing difficulty accompanied by a remarkable performance decline.Biohybrid robots driven by living cells can be a potential solution to overcome these drawbacks by benefiting from the intrinsic microscale self-assembly of living tissues and high energy efficiency,which,among other unprecedented properties,also feature flexibility,self-repair,and even multiple degrees of freedom.This paper systematically reviews the development of biohybrid robots.First,the development of biological flexible drivers is introduced while emphasizing on their advantages over traditional drivers.Second,up-to-date works regarding biohybrid robots are reviewed in detail from three aspects:biological driving sources,actuator materials,and structures with associated control methodologies.Finally,the potential future applications and major challenges of biohybrid robots are explored.

Interaction between Bax and Bcl-XL proteins confirmed by partial acceptor photobleaching-based FRET imaging

Exact interaction mechanism between Bax and Bcl-XL,two key Bcl-2 family proteins,is an interesting and controversial issue.Partial acceptor photobleaching-based quantitative fluores-cence resonance energy transfer(FRET)measurement,PbFRET,is a widely used FRET quantification method in living cells.In this report,we implemented pixel-to-pixel PbFRET imaging on a wide-field microscope to map the FRET efficiency(E)images of single living HepG2 cells co-expressing CFP-Bax and YFP-Bcl-XL.The E value between CFP-Bax and YFP-Bcl-XL was 4.59%in cytosol and 11.31%on mitochondria,conclusively indicating the direct interaction of the two proteins,and the interaction of the two proteins was strong on mitochondria and modest in cytosol.

Highly-efficient quantitative fluorescence resonance energy transfer measurements based on deep learning

Intensity-based quantitative fluorescence resonance energy transfer(FRET)is a technique to measure the distance of molecules in scale of a few nanometers which is far beyond optical diffraction limit.This widely used technique needs complicated experimental process and manual image analyses to obtain precise results,which take a long time and restrict the application of quantitative FRET especially in living cells.In this paper,a simplified and automatic quanti-tative FRET(saqFRET)method with high efficiency is presented.In saqFRET,photo-activatable acceptor PA-mCherry and optimized excitation wavelength of donor enhanced green fluorescent protein(EGFP)are used to simplify FRET crosstalk elimination.Traditional manual image analyses are time consuming when the dataset is large.The proposed automatic image analyses based on deep learning can analyze 100 samples within 30 s and demonstrate the same precision as manual image analyses.

Green emitted CdSe@ZnS quantum dots for FLIM and STED imaging applications

Inorganic quantum dots(QDs)have excellent optical properties,such as high°uorescence intensity,excellent photostability and tunable emission wavelength,etc.,facilitating them to be used as labels and probes for bioimaging.In this study,CdSe@ZnS QDs are used as probes for Fluorescence lifetime imaging microscope(FLIM)and stimulated emission depletion(STED)nanoscopy imaging.The emission peak of CdSe@ZnS QDs centered at 526 nm with a narrow width of 19 nm and the photoluminescence quantum yield(PLQY)was 64%.The QDs presented excellent anti-photobleaching property which can be irradiated for 400 min by STED laser with 39.8 mW.The lateral resolution of 42.0 nm is demonstrated for single QDs under STED laser(27.5 mW)irradiation.Furthermore,the CdSe@ZnS QDs were for the first time used to successfully label the lysosomes of living HeLa cells and 81.5 nm lateral resolution is obtained indicating the available super-resolution applications in living cells for inorganic QD probes.Meanwhile,Eca-109 cells labeled with the CdSe@ZnS QDs was observed with FLIM,and their fluorescence lifetime was around 3.1 ns,consistent with the in vitro value,suggesting that the QDs could act as a satisfactory probe in further FLIM-STED experiments.

QUANTITATIVE FRET MEASUREMENT BASED ON CONFOCAL MICROSCOPY IMAGING AND PARTIAL ACCEPTOR PHOTOBLEACHING

Fluorescence resonance energy transfer(FRET)technology had been widely used to study protein
protein interactions in living cells.In this study,we developed a ROI-PbFRET method to real-time quantitate the FRET efficiency of FRET construct in living cells by combining the region of interest(ROI)function of confocal microscope and partial acceptor photobleaching.We validated the ROI-PbFRET method using GFPs-based FRET constructs including 18AA and SCAT3,and used it to quantitatively monitor the dynamics of caspase-3 activation in single live cells stably expressing SCAT3 during staurosporine(STS)-induced apoptosis.Our results for thefirst demonstrate that ROI-PbFRET method is a powerful potential tool for detecting the dynamics of molecular interactions in live cells.

Ultra-thin temperature controllable microwell array chip for continuous real-time high-resolution imaging of living single cells

Single-cell imaging,a powerful analytical method to study single-cell behavior,such as gene expression and protein profiling,provides an essential basis for modern medical diagnosis.The coding and localization function of microfluidic chips has been developed and applied in living single-cell imaging in recent years.Simultaneously,chip-based living single-cell imaging is also limited by complicated trapping steps,low cell utilization,and difficult high-resolution imaging.To solve these problems,an ultra-thin temperature-controllable microwell array chip(UTCMA chip)was designed to develop a living single-cell workstation in this study for continuous on-chip culture and real-time high-resolution imaging of living single cells.The chip-based on ultra-thin ITO glass is highly matched with an inverted microscope(or confocal microscope)with a high magnification objective(100×oil lens),and the temperature of the chip can be controlled by combining it with a home-made temperature control device.High-throughput single-cell patterning is realized in one step when the microwell array on the chip uses hydrophilic glass as the substrate and hydrophobic SU-8 photoresist as the wall.The cell utilization rate,single-cell capture rate,and microwell occupancy rate are all close to 100%in the microwell array.This method will be useful in rare single-cell research,extending its application in the biological and medical-related fields,such as early diagnosis of disease,personalized therapy,and research-based on single-cell analysis.

Water-soluble Cu30 nanoclusters as a click chemistry catalyst for living cell labeling via azide-alkyne cycloaddition

Cu(I)-catalyzed azide-alkyne cycloadditions(CuAAC)have gained increasing interest in the selective labeling of living cells and organisms with biomolecules.However,their application is constrained either by the high cytotoxicity of Cu(I)ions or the low activity of CuAAC in the internal space of living cells.This paper reports the design of a novel Cu-based nanocatalyst,watersoluble thiolated Cu30 nanoclusters(NCs),for living cell labeling via CuAAC.The Cu30 NCs offer good biocompatibility,excellent stability,and scalable synthesis(e.g.,gram scale),which would facilitate potential commercial applications.By combining the highly localized Cu(I)active species on the NC surface and good structural stability,the Cu30 NCs exhibit superior catalytic activities for a series of Huisgen cycloaddition reactions with good recyclability.More importantly,the biocompatibility of the Cu30 NCs enables them to be a good catalyst for CuAAC,whereby the challenging labeling of living cells can be achieved via CuAAC on the cell membrane.This study sheds light on the facile synthesis of atomically precise Cu NCs,as well as the design of novel Cu NCs-based nanocatalysts for CuAAC in intracellular bioorthogonal applications.

Effect of Probe Lifting Height in Jumping Mode AFM for Living Cell Imaging

Atomic force microscopy(AFM)is one of the effective methods for imaging the morphological and physical properties of living cells in a near-physiological environment.However,several problems caused by the adhesion of living cells and extension of the cell membranes seriously affect the image quality during living cell imaging,hindering the study of living cells.In this work,jumping mode AFM imaging was used to image living cells at varied probe lifting heights to meet image quality requirements,and image quality related to the probe lifting height is discussed in detail.The jumping mode was divided into three parts based on the varying heights of the lifted probe,namely near-contact mode,half-jumping mode,and full-jumping mode,and the causes of their imaging drawbacks were analyzed.At an appropriate lifting height,the probe can be completely free from the influence of cell adhesion and self-excited oscillation,thus avoiding the occurrence of“trail”phenomena and invalid points in the imaging of living cells and improving the image quality.Additionally,this work provides a new approach to calculating the lateral force through the adhesion of trace and retrace scanning at a low height,which is important for studying the extension characteristics of the cell membrane.

Recent advances in small molecule fluorescent probes for simultaneous imaging of two bioactive molecules in live cells and in vivo

The interrelationships and synergistic regulations of bioactive molecules play pivotal roles in physiological and pathological processes involved in the initiation and development of some diseases,such as cancer and neurodegenerative and cardiovascular diseases.Therefore,the simultaneous,accurate and timely detection of two bioactive molecules is crucial to explore their roles and pathological mechanisms in related diseases.Fluorescence imaging associated with small molecular probes has been widely used in the imaging of bioactive molecules in living cells and in vivo due to its excellent performances,including high sensitivity and selectivity,noninvasive properties,real-time and high spatial temporal resolution.Single organic molecule fluorescent probes have been successively developed to simultaneously monitor two biomolecules to uncover their synergistic relationships in living systems.Hence,in this review,we focus on summarizing the design strategies,classifications,and bioimaging applications of dual-response fluorescent probes over the past decade.Furthermore,future research directions in this field are proposed.

Living cell for drug delivery

The living cells have been emerged as useful platforms for drug delivery due to their advantages of good liquidity,stability,and low immunogenicity.In this review,we summarized the development of living cells-based drug delivery systems.The drug loading methods,applications,and advantages of living cell drug delivery systems were summarized.Different living cells for drug delivery,the mechanisms of action,therapeutic applications,as well as main features were summarized and highlighted.The recent research progress and challenges were discussed.The future directions and prospects were proposed.