Lysosomes Initiating and DNAzyme-Assisted Intracellular Signal Amplification Strategy for Quantification of Alpha-Fetoprotein in a Single Cell.

Cellular trace proteins are critical for maintaining normal cell functions, with their quantitative analysis in individual cells aiding our understanding of the role of cell proteins in biological processes. This study proposes a strategy for the quantitative analysis of alpha-fetoprotein in single cells, utilizing a lysosome microenvironment initiation and a DNAzyme-assisted intracellular signal amplification technique based on electrophoretic separation. A nanoprobe targeting lysosomes was prepared, facilitating the intracellular signal amplification of alpha-fetoprotein. Following intracellular signal amplification, the levels of alpha-fetoprotein (AFP) in 20 HepG2 hepatoma cells and 20 normal HL-7702 hepatocytes were individually evaluated using microchip electrophoresis with laser-induced fluorescence detection (MCE-LIF). Results demonstrated overexpression of alpha-fetoprotein in hepatocellular carcinoma cells. This strategy represents a novel technique for single-cell protein analysis and holds significant potential as a powerful tool for such analyses.

Proximity-Induced Bipedal DNA Walker for Accurately Visualizing microRNA in Living Cancer Cell.

DNA walker, a type of dynamic DNA device that is capable of moving progressively along prescribed walking tracks, has emerged as an ideal and powerful tool for biosensing and bioimaging. However, most of the reported three-dimensional (3D) DNA walker were merely designed for the detection of a single target, and they were not capable of achieving universal applicability. Herein, we reported for the first time the development of a proximity-induced 3D bipedal DNA walker for imaging of low abundance biomolecules. As a proof of concept, miRNA-34a, a biomarker of breast cancer, is chosen as the model system to demonstrate this approach. In our design, the 3D bipedal DNA walker can be generated only by the specific recognition of two proximity probes for miRNA-34a. Meanwhile, it stochastically and autonomously traveled on 3D tracks (gold nanoparticles) via catalytic hairpin assembly (CHA), resulting in the amplified fluorescence signal. In comparison with some conventional DNA walkers that were utilized for living cell imaging, the 3D DNA walkers induced by proximity ligation assay can greatly improve and ensure the high selectivity of bioanalysis. By taking advantage of these unique features, the proximity-induced 3D bipedal DNA walker successfully realizes accurate and effective monitoring of target miRNA-34a expression levels in living cells, affording a universal, valuable, and promising platform for low-abundance cancer biomarker detection and accurate identification of cancer.

A Macrophage Membrane-Coated Cu-WO3-x-Hydro820 Nanoreactor for Treatment and Photoacoustic/Fluorescence Dual-Mode Imaging of Inflamed Liver Tissue.

A disease-targeting nanoplatform that integrates imaging with therapeutic activity would facilitate early diagnosis, treatment, and therapeutic monitoring. To this end, a macrophage membrane-coated Cu-WO3-x-Hydro820 (CWHM) nanoreactor was prepared. This reactor was shown to target inflammatory tissues. The reactive oxygen species (ROS) such as H2O2 and ·OH in inflammatory tissues can react with Hydro820 in the reactor to form the NIR fluorophore IR820. This process allowed photoacoustic/fluorescence dual-mode imaging of H2O2 and ·OH, and it is expected to permit visual diagnosis of inflammatory diseases. The Cu-WO3-x nanoparticles within the nanoreactor shown catalase and superoxide enzyme mimetic activity, allowing the nanoreactor to catalyze the decomposition of H2O2 and ·O2- in inflammatory cells of hepatic tissues in a mouse model of liver injury, thus alleviating the oxidative stress of damaged liver tissue. This nanoreactor illustrates a new strategy for the diagnosis and treatment of hepatitis and inflammatory liver injury.

Acid-Triggered Degradation of Three-In-One Ag2S Quantum Dots for In Situ Ratiometric NIR-II Fluorescence Imaging-Guided Ion/Gas Combination Therapy.

Smart theranostic nanoprobes with the integration of multiple therapeutic modalities are preferred for precise diagnosis and efficient therapy of tumors. However, it remains a big challenge to arrange the imaging and two or more kinds of therapeutic agents without weakening the intended performances. In addition, most existing fluorescence (FL) imaging agents suffer from low spatiotemporal resolution due to the short emission wavelength (<900 nm). Here, novel three-in-one Ag2S quantum dot (QD)-based smart theranostic nanoprobes were proposed for in situ ratiometric NIR-II FL imaging-guided ion/gas combination therapy of tumors. Under the acidic tumor microenvironment, three-in-one Ag2S QDs underwent destructive degradation, generating toxic Ag+ and H2S. Meanwhile, their FL emission at 1270 nm was weakened. Upon introduction of a downconversion nanoparticle (DCNP) as the delivery carrier and NIR-II FL reference signal unit, the formed Ag2S QD-based theranostic nanoprobes could achieve precise diagnosis of tumors through ratiometric NIR-II FL signals. Also, the generated Ag+ and H2S enabled specific ion/gas combination therapy toward tumors. By combining the imaging and therapeutic functions, three-in-one Ag2S QDs may open a simple yet reliable avenue to design theranostic nanoprobes.

Improving the sensing sensitivity of silver nanoparticle-based colorimetric biosensors from the point of salt.

The sensing sensitivity was improved for silver nanoparticles (AgNPs)-based colorimetric biosensors by using the most suitable salt to induce AgNPs aggregation. As for the salt composed of low-affinity anion and monovalent cation, the cation-dependent charge screening effect was the driving force for AgNPs aggregation. Apart from the charge screening effect, both the bridging of multivalent cation to the surface ligand of AgNP and the interaction between anion and Ag contributed to inducing AgNPs aggregation. Considering the higher aggregation efficiency of AgNPs resulted in a narrower sensing range, salt composed of low-affinity anion and monovalent cation was recommended for AgNPs-based colorimetric analysis, which was confirmed by fourfold higher sensitivity of DNA-21 detection using NaF than NaCl. This work inspires further thinking on improving the sensing performance of metal nanomaterials-based sensors from the point of colloidal surface science.

A Framework Nucleic Acid-Mediated Multimodal Tandem Multivariate Signal Amplification Strategy for Simultaneous Absolute Quantification of Three Different MicroRNAs in a Single Cell.

The simultaneous quantification of multiple microRNAs (miRNA) in a single cell can help scientists understand the relationship between different miRNA groups and different types of cancers from an miRNA omics perspective at the single-cell level. However, there currently remains a challenge in developing techniques for the simultaneous absolute quantification of multiple miRNAs in single cells. Herein, we propose a framework nucleic acid (FNA)-mediated multimodal tandem multivariate signal amplification strategy for simultaneous absolute quantification of three different miRNAs in a single cell. In this study, DNA hexahedron FNAs (DHFs) and DNA tetrahedron FNAs (DTFs) were first prepared, multiple DNA hairpins and substrates were then connected to the hexahedron frame nucleic acid as the target recognition units, and three substrates with labeled FAM fluorophores on the tetrahedral frame nucleic acid served as signal output units. After the two types of FNAs entered the cell, they reacted with three different miRNAs (miRNA-155, miRNA-373, and miRNA-21) and multimodal tandem multivariate signal amplification was initiated simultaneously, reducing the detection limit of the three miRNAs to 8 × 10-15, 2 × 10-15, and 1 × 10-15 M, respectively. The detection sensitivity of the three miRNAs was simultaneously increased by six orders of magnitude, reaching the quantitative requirement of trace miRNAs in single cells. Combined with single-cell injection, membrane melting, and intracellular component separation technology on a microchip electrophoresis platform, we achieved the simultaneous absolute quantification of three different miRNAs in a single cell, thereby providing an important novel method that can be used to conduct single-cell research.

Single-Excitation Triple-Emission Down-/Up-Conversion Nanoassemblies for Tumor Microenvironment-Enhanced Ratiometric NIR-II Fluorescence Imaging and Chemo-/Photodynamic Combination Therapy.

Tumor microenvironment-mediated ratiometric second near-infrared (NIR-II) fluorescence imaging and photodynamic therapy contribute to accurate diagnosis and highly efficient therapy of deep tumors. However, it is challenging to integrate these functions into one nanodrug due to the difficulty in preparing triple-emission nanoprobes. In this work, single-excitation triple-emission (wavelength at 660, 1060, and 1550 nm) down-/up-conversion nanoassemblies were prepared by conjugating dual-ligands-stabilized gold nanoclusters (cgAuNCs) into down-/up-conversion nanoparticles (D/UCNPs), which simultaneously realized ratiometric NIR-II fluorescence imaging and chemo-/photodynamic combination therapy toward tumors upon exposure to an 808 nm laser. The presence of dual ligands endowed cgAuNCs with an enhanced NIR-II fluorescence response to endogenous glutathione, allowing in situ ratiometric NIR-II fluorescence imaging of tumors using the prepared nanoassemblies. Additionally, the stabilizing ligand cyclodextrin of cgAuNCs facilitated the loading of the antitumor drug doxorubicin, and D/UCNPs could be modified with the photosensitizer methylene blue. Such a spatially separated functionalization method enabled chemo-/photodynamic combination therapy. This study provides new insights into the design of multifunctional nanoplatforms for tumor diagnosis and treatment.

Engineered Cancer Cells as Signal Probes for Fluorescence-Assisted Digital Counting Analysis.

Fluorescence-assisted digital counting analysis allowed sensitive quantification of targets by measuring individual fluorescent labels. However, traditional fluorescent labels suffered from low brightness, small size, and sophisticated preparation procedures. Herein, engineering fluorescent dye-stained cancer cells with magnetic nanoparticles were proposed to construct single-cell probes for fluorescence-assisted digital counting analysis by quantifying the target-dependent binding or cleaving events. Various engineering strategies of cancer cells including biological recognition and chemical modification were developed for rationally designing single-cell probes. Introduction of suitable recognition elements into single-cell probes allowed digital quantification of each target-dependent event via counting the colored single-cell probes in the representative image taken using a confocal microscope. The reliability of the proposed digital counting strategy was corroborated by traditional optical microscopy- and flow cytometry-dependent counting technologies. The advantages of single-cell probes, including high brightness, big size, ease of preparation, and magnetic separation, contributed to the sensitive and selective analysis of targets of interest. As proof-to-concept assays, indirect analysis of exonuclease III (Exo III) activity, as well as direct quantitation of cancer cells, were investigated, and the potential in biological sample analysis was also assessed. This sensing strategy will open a new avenue for the development of biosensors.

Fluid Multivalent Recognition Accelerating and Boosting Upconversion Luminescence-Activated DNA Nanomachines for Rapid and Sensitive In Vivo Imaging.

By integrating near-infrared (NIR) light-dependent optical control and DNA walkers-based signal amplification, upconversion luminescence-activated DNA nanomachines hold great potential in conducting an in vivo analysis. For the typical DNA nanomachines, the immobile multivalent recognition interface greatly compromised the reaction kinetics and amplification efficiency due to the cleavage-dependent response mode. In this work, novel upconversion luminescence-activated DNA nanomachines with a fluid multivalent recognition interface were reported for rapid and sensitive in vivo imaging. As a proof-of-concept study, the photolocked DNAzyme-based walker system was anchored on the surface of phospholipid membrane-coated upconversion nanoparticles through the cholesterol-phospholipid interaction to acquire a fluid multivalent recognition interface. Upon sequential inputs of NIR light and metal ions, the formed DNA nanomachines were autonomously initiated and generated a cascade of amplified signal. Relative to the typical DNA nanomachines, the proposed ones possess an accelerated reaction rate and an improved amplification capability owing to a higher local concentration by the lateral mobility. The present work provides a versatile alternative for performing precise and highly efficient in vivo analysis.

Fluidic Membrane Accelerating the Kinetics of Photoactivatable Hybridization Chain Reaction for Accurate Imaging of Tumor-Derived Exosomes.

Slow intermolecular collisions and "always active" responses compromise the amplification efficiency and response accuracy of nonenzymatic hybridization chain reaction (HCR). In this study, a photoactivatable membrane-oriented HCR (MOHCR) system was rationally designed by binding a photocleavable initiator probe onto a target protein and then anchoring cholesterol-modified hairpin-structure fuel probes. When irradiated, the bound initiator probe was photoactivated and initiated self-assembly to generate activatable and amplified imaging. In a proof-of-concept assay, breast-cancer-derived exosomes were imaged based on the surface protein epithelial cell adhesion molecule (EpCAM). Photoactivatable responses provided precise spatiotemporal control of the MOHCR, and fluidic membranes enabled accelerated reaction kinetics. Our MOHCR system demonstrated high efficiency and accuracy in differentiating between plasma samples from breast cancer patients and healthy donors.

Ultraviolet C Irradiation-Induced Dehybridization of Double-Stranded Oligonucleotides: Mechanism Investigation and Label-Free Measurement of the Photodamage Level.

Elucidating the mechanism and estimating the extent of conformation change of double-stranded DNA (dsDNA) upon ultraviolet (UV) exposure are of vital importance for understanding the DNA photodamage process. The existing research was mainly focused on the lesions of single-stranded DNA (ssDNA) and involved off-site measurement of the photodamage level. In this work, short-wavelength UV (UVC) (254 nm) irradiation was demonstrated to induce the dehybridization of dsDNA due to the loss of paring capacity of photodamaged pyrimidine nucleobases. The intrinsic programmability of dsDNA enabled researchers to rationally design the on-demand dehybridization sites. The spatial conformation switch of dsDNA caused by UVC irradiation could be evolved into a label-free sensing platform for the on-site measurement of the DNA photodamage level.

A gas-pressure-assisted ratiometric atomic flame assay for the point-of-care testing of tumor-cell-derived exosomes.

The multicolor-based point-of-care testing (POCT) of tumor cell-derived exosomes is of vital importance for understanding tumor growth and metastasis. Multicolor-based ratiometric signals most often rely on molecular optics, such as fluorescence resonance energy transfer (FRET)-dependent molecular fluorescence and localized surface plasmon resonance (LSPR)-related molecular colorimetry. However, finding acceptable FRET donor-acceptor fluorophore pairs and the kinetically slow color responses during size-related molecular colorimetry have greatly impeded POCT applications. Herein, an atomic flame was used to develop a visual sensing platform for the POCT of tumor-cell-derived exosomes. In comparison with common molecular optics, the atomic flame possessed the advantages of providing both a variety of ratiometric flame signals and fast response sensitivity. The integration of a gas-pressure-assisted flame reaction and dual-aptamer recognition guaranteed the sensitive and selective analysis of exosomes with a low limit of detection (LOD) of 7.6 × 102 particles per mL. Such a novel optical signal will inspire the development of more user-friendly POCT approaches.

Electrochemical determination of caspase-3 using signal amplification by HeLa cells modified with silver nanoparticles.

An electrochemical sensor capable of quantitative determination of caspase-3 activities was developed. A thiolated peptide whose sequence contained a caspase-3 cleaved site and a cell penetration sequence was preimmobilized onto an electrode. The quantification of caspase-3 was accomplished after cell penetration and the subsequent adsorption of silver nanoparticles (AgNPs). The oxidation current of AgNPs was found to be inversely proportional to the concentration of caspase-3 between 0.02 and 0.2 U/mL. A detection limit of 0.02 U/mL for caspase-3 was achieved due to the large number of positively charged AgNPs adsorbed onto the negatively charged cells. The proof of concept was demonstrated by monitoring the cleavage of surface-confined peptide substrates by caspase-3 in cell lysates. The current sensor could be extended to detect cells by replacing the surface-confined peptide with aptamers that recognize cells. Thus, the use of a cell as a matrix for AgNPs shows excellent potential for constructing electrochemical sensors and provides a useful alternative for sensor development in the future. Cells modified with silver nanoparticles were utilized as the electrochemical readout of an electrochemical assay.

Dissecting the Effect of Salt for More Sensitive Label-Free Colorimetric Detection of DNA Using Gold Nanoparticles.

Taking advantage of the protection effect of single-stranded DNA oligonucleotides, gold nanoparticles (AuNPs) remain dispersed and retain a red color with the addition of a low concentration of salt, while AuNPs would aggregate in the presence of double-stranded DNA. This difference has been used to design label-free colorimetric sensors for DNA detection. NaCl is the most commonly used salt to induce the aggregation of AuNPs. In this work, we aimed to test if other salts can provide even better sensor performance and to understand the effects of the cations and anions in salts. We first studied the effect of anions, including halides (NaF, NaCl, NaBr, and NaI), and other common salts (NaNO3, NaClO4, Na2SO4, Na2S2O3, sodium phosphate, and sodium citrate). Among them, weakly adsorbing ones such as F-, citrate, and phosphate appeared to yield better sensitivity than Cl-. Anions can directly adsorb on the AuNPs and affect DNA adsorption. We then tested cations, and only group 1A metals (LiCl, NaCl, KCl, RbCl, and CsCl) can signal DNA adsorption, while divalent metals (MgCl2, CaCl2, MnCl2, and NiCl2) barely showed the effect of DNA. CsCl only works for strongly adsorbing DNA, such as A15, but not weakly adsorbing T15. Overall, NaF is a better salt than NaCl by having a 2.3-fold higher sensitivity, which was confirmed in a DNA sensing assay. This work has identified a better salt yielding higher sensitivity, and sensing work relying on the change of the aggregation state of AuNPs can benefit from this study.

NIR Light-Responsive Hollow Porous Gold Nanospheres for Controllable Pressure-Based Sensing and Photothermal Therapy of Cancer Cells.

Pressure-based signal transduction has attracted recent and extensive attention due to its high sensitivity and simplicity. The most popular way to generate gas pressure relies on catalyst-mediated decomposition of H2O2. Despite its high sensitivity, this method lacks spatial and temporal control of the reaction, and may suffer from variations due to the dead time of mixing. In this work, we report a new reaction using near-infrared (NIR) light to heat hollow porous gold nanospheres (AuNSs) for thermal decomposition of NH4HCO3. Comparisons were made on these two systems especially on controlled pressure production. As an example of application, our light-controlled system was used for the detection of MCF-7 cancer cells by attaching the S2.2 aptamer on the AuNSs. The detection limit was as low as 2 cells/mL. Meanwhile, the heat produced by the AuNSs was used to induce localized hyperthermia at the surface of the cancer cells. This interesting theranostic system provides new insights into pressure-based sensing and may inspire new analytical applications.

Sensitive and simultaneous surface plasmon resonance detection of free and p53-bound MDM2 proteins from human sarcomas.

Murine double minute 2 (MDM2) is an oncoprotein mediating the degradation of the tumor suppressor p53 protein. The physiological levels of MDM2 protein are closely related to malignant transformation and tumor growth. In this work, the simultaneous and label-free determination of free and p53-bound MDM2 proteins from sarcoma tissue extracts was conducted using a dual-channel surface plasmon resonance (SPR) instrument. Free MDM2 protein was measured in one fluidic channel covered with the consensus double-stranded (ds)-DNA/p53 conjugate, while MDM2 bound to p53 was captured by the consensus ds-DNA immobilized onto the other channel. To achieve higher sensitivity and to confirm specificity, an MDM2-specific monoclonal antibody (2A10) was used to recognize both the free and p53-bound MDM2 proteins. The resultant method afforded a detection limit of 0.55 pM of MDM2. The amenability of the method to the analysis of free and p53-bound MDM2 proteins was demonstrated for normal and sarcoma tissue extracts from three patients. Our data reveal that both free and total MDM2 (free and bound forms combined) proteins from sarcoma tissue extracts are of much higher concentrations than those from normal tissue extracts and the p53-bound MDM2 protein only constitutes a small fraction of the total MDM2 concentration. In comparison with enzyme-linked immunosorbent assay (ELISA), the proposed method possesses higher sensitivity, is more cost-effective, and is capable of determining free and p53-bound MDM2 proteins in clinical samples.

Sensitive Hg2+ Sensing via Quenching the Fluorescence of the Complex between Polythymine and 5,10,15,20-tetrakis(N-methyl-4-pyridyl) Porphyrin (TMPyP).

The interaction between polythymine (dTn) and 5,10,15,20-tetrakis(N-methyl-4-pyridyl) porphyrin (TMPyP) was systematically studied using various techniques. dTn remarkably enhanced the fluorescence intensity of TMPyP as compared to other oligonucleotides. The enhanced fluorescence intensity and the shift of the emission peaks were ascribed to the formation of a π-π complex between TMPyP and dTn. And the quenching of the dTn-enhanced fluorescence by Hg2+ through a synergistic effect occurs due to the heavy atom effect. The binding of Hg2+ to TMPyP plays an important role in the Hg-TMPyP-dT30 ternary complex formation. A TMPyP-dT30-based Hg2+ sensor was developed with a dynamic range of Hg2+ from 5 nM to 100 nM. The detection limit of 1.3 nM was low enough for Hg2+ determination. The sensor also exhibited good selectivity against other metal ions. Experiments for tap water and river water demonstrated that the detection method was applicable for Hg2+ determination in real samples. The Hg2+ sensor based on oligonucleotide dT30-enhanced TMPyP fluorescence was fast and low-cost, presenting a promising platform for practical Hg2+ determination.

DNA Generated Electric Current Biosensor.

In addition to its primary function as a genetic material, deoxyribonucleic acid (DNA) is also a potential biologic energy source for molecular electronics. For the first time, we demonstrate that DNA can generate a redox electric current. As an example of this new functionality, DNA generated redox current was used for electrochemical detection of human epidermal growth factor receptor 2 (HER2), a clinically important breast cancer biomarker. To induce redox current, the phosphate of the single stranded DNA aptamer backbone was reacted with molybdate to form redox molybdophosphate precipitate and generate an electrochemical current of ∼16.8 µA/µM cm2. This detection of HER2 was performed using a sandwich detection assay. A HER2 specific peptide was immobilized onto a gold electrode surface for capturing HER2 in buffer and serum. The HER2 specific aptamer was used as both ligand to bind the captured HER2 and to generate a redox current signal. When tested for HER2 detection, the electrochemical current generated by the aptasensor was proportional to HER2 concentration in the range of 0.01 to 5 ng/mL, with a current generated in the range of ∼6.37 to 31.8 µA/cm2 in both buffer and serum. This detection level is within the clinically relevant range of HER2 concentrations. This method of electrochemical signal amplification greatly simplifies the signal transduction of aptasensors, broadening their use for HER2 analysis. This novel approach of using the same aptamer as biosensor ligand and as transducer can be universally extended to other aptasensors for a wide array of biodetection applications. Moreover, electric currents generated by DNA or other nucleic acids can be used in molecular electronics or implanted devices for both power generation and measurement of output.

Surface plasmon resonance biosensors for simultaneous monitoring of amyloid-beta oligomers and fibrils and screening of select modulators.

Oligomeric amyloid-beta (Aß) peptides are considered as the most toxic species in Alzheimer's disease (AD). Monitoring of the Aß aggregation profiles is critical for elucidating the oligomer toxicity and may serve as a therapeutic target for AD. By immobilizing the capture antibodies of A11 and OC that are specific to the oligomers and fibrils, respectively, in separate fluidic channels, a novel surface plasmon resonance (SPR) biosensor was designed for monitoring the oligomeric and fibrillar species of Aß(1-42) simultaneously. The influence of curcumin, Cu(2+) and methylene blue on the amount of toxic oligomers and fibrils was evaluated. The half maximal inhibitory concentration (IC50) of curcumin and methylene blue was determined. The formation of Aß fibrils was also validated by the thioflavin T (ThT) fluorescence assay. The results demonstrate the utility of SPR as an analytical tool for rapid and comprehensive monitoring of Aß aggregation and screening of Aß modulators.

Silver nanoclusters-based fluorescence assay of protein kinase activity and inhibition.

A simple and sensitive fluorescence method for monitoring the activity and inhibition of protein kinase (PKA) has been developed using polycytosine oligonucleotide (dC12)-templated silver nanoclusters (Ag NCs). Adenosine-5'-triphosphate (ATP) was found to enhance the fluorescence of Ag NCs, while the hydrolysis of ATP to adenosine diphosphate (ADP) by PKA decreased the fluorescence of Ag NCs. Compared to the existing methods for kinase activity assay, the developed method does not involve phosphorylation of the substrate peptides, which significantly simplifies the detection procedures. The method exhibits high sensitivity, good selectivity, and wide linear range toward PKA detection. The inhibition effect of kinase inhibitor H-89 on the activity of PKA was also studied. The sensing protocol was also applied to the assay of drug-stimulated activation of PKA in HeLa cell lysates.