Construction of AIEgens-Based Bioprobe with Two Fluorescent Signals for Enhanced Monitor of Extracellular and Intracellular Telomerase Activity

Detections of telomerase activity in vitro and in living cells are of great importance for clinical diagnosis of cancer. In this work, an AIEgens-based bioprobe with two fluorescent signals for enhanced monitor of extracellular and intracellular telomerase activity is designed. After addition of telomerase, two positively charged AIEgens (Silole-R and TPE-H) bind to quencher group labeled primer (QP) and the extension repeated units, leading enhancement of two telomerase-triggered fluorescent signals. Furthermore, by combination the wider linear range in vitro and lower background in living cells imaging, the bioprobe is used to detect telomerase extracted from various cell lines (MCF-7, HeLa, E-J, and HLF), 50 bladder cancer patients’ urine samples, 10 normal people’s urine samples, and also applied in mapping telomerase activity inside living cells (MCF-7, HeLa, MDA-MB-231, and HT1080). The results show that this well-designed strategy can successfully detect telomerase activity in vitro and in living cells with high sensitivity, indicating the potential application of this method in cancer cells bioimaging and clinical cancer diagnosis

Highly-Efficient Gating of Solid-State Nanochannels by DNA Supersandwich Structure Containing ATP Aptamers: A Nanofluidic IMPLICATION Logic Device

Integrating biological components into artificial devices establishes an interface to understand and imitate the superior functionalities of the living systems. One challenge in developing biohybrid nanosystems mimicking the gating function of the biological ion channels is to enhance the gating efficiency of the man-made systems. Herein, we demonstrate a DNA supersandwich and ATP gated nanofluidic device that exhibits high ON–OFF ratios (up to 10⁶) and a perfect electric seal at its closed state (∼GΩ). The ON–OFF ratio is distinctly higher than existing chemically modified nanofluidic gating systems. The gigaohm seal is comparable with that required in ion channel electrophysiological recording and some lipid bilayer-coated nanopore sensors. The gating function is implemented by self-assembling DNA supersandwich structures into solid-state nanochannels (open-to-closed) and their disassembly through ATP–DNA binding interactions (closed-to-open). On the basis of the reversible and all-or-none electrochemical switching properties, we further achieve the IMPLICATION logic operations within the nanofluidic structures. The present biohybrid nanofluidic device translates molecular events into electrical signals and indicates a built-in signal amplification mechanism for future nanofluidic biosensing and modular DNA computing on solid-state substrates

Bioelectrochemical Switches for the Quantitative Detection of Antibodies Directly in Whole Blood

The development of rapid, low-cost point-of-care approaches for the quantitative detection of antibodies would drastically impact global health by shortening the delay between sample collection and diagnosis and by improving the penetration of modern diagnostics into the developing world. Unfortunately, however, current methods for the quantitative detection of antibodies, including ELISAs, Western blots, and fluorescence polarization assays, are complex, multiple-step processes that rely on well-trained technicians working in well-equipped laboratories. In response, we describe here a versatile, DNA-based electrochemical “switch” for the rapid, single-step measurement of specific antibodies directly in undiluted whole blood at clinically relevant low-nanomolar concentrations

Facile Probe Design: Fluorescent Amphiphilic Nucleic Acid Probes without Quencher Providing Telomerase Activity Imaging Inside Living Cells

Nowadays, the probe with fluorophore but no quencher is promising for its simple preparation, environmental friendliness, and wide application scope. This study designs a new amphiphilic nucleic acid probe (ANAP) based on aggregation-caused quenching (ACQ) effect without any quencher. Upon binding with targets, the dispersion of hydrophobic part (conjugated fluorene, CF) in ANAP is enhanced as a signal-on model for proteins, nucleic acids, and small molecules detection or the aggregation of CF is enhanced as a signal-off model for ion detection. Meanwhile, because of the high specificity of ANAP, a one-step method is developed powerfully for monitoring the telomerase activity not only from the cell extracts but also from 50 clinic urine samples (positive results from 45 patients with bladder cancer and negative results from 5 healthy people). ANAPs can also readily enter into cells and exhibit a good performance for distinguishing natural tumor cells from the tumor cells pretreated by telomerase-related drugs or normal cells. In contrast to our previous results (Anal. Chem. 2015, 87, 3890−3894), the present CF is a monomer which is just the structure unit of the previous fluorescent polymer. Since the accurate molecular structure and high DNA/CF ratio of the present CF, these advanced experiments obtain an easier preparation of probes, an improved sensitivity and specificity, and broader detectable targets

Facile, Fast-Responsive, and Photostable Imaging of Telomerase Activity in Living Cells with a Fluorescence Turn-On Manner

In situ detecting and monitoring intracellular telomerase activity is significant for cancer diagnosis. In this work, we report a facile and fast-responsive bioprobe for in situ detection and imaging of intracellular telomerase activity with superior photostability. After transfected into living cells, quencher group labeled TS primer (QP) can be extended in the presence of intracellular telomerase. Positive charged TPE-Py molecules (AIE dye) will bind to the primer as well as extension repeated units, producing a telomerase activity-related turn-on fluorescence signal. By incorporating positive charged AIE dye and substrate oligonucleotides, in situ light-up imaging and detection of intracellular telomerase activity were achieved. This strategy exhibits good performance for sensitive in situ tracking of telomerase activity in living cells. The practicality of this facile and fast-responsive telomerase detection method was demonstrated by using it to distinguish tumor cells from normal cells and to monitor the change of telomerase activity during treatment with antitumor drugs, which shows its potential in clinical diagnostic and therapeutic monitoring

Photoactivated Specific mRNA Detection in Single Living Cells by Coupling “Signal-on” Fluorescence and “Signal-off” Electrochemical Signals

The spatiotemporal detection of a target mRNA in a single living cell is a major challenge in nanoscience and nanomedicine. We introduce a versatile method to detect mRNA at a single living cell level that uses photocleavable hairpin probes as functional units for the optical (fluorescent) and electrochemical (voltammetric) detection of MnSOD mRNA in single MCF-7 cancer cells. The fluorescent probe is composed of an ortho-nitrophenylphosphate ester functionalized hairpin that includes the FAM fluorophore in a caged configuration quenched by Dabcyl. The fluorescent probe is further modified with the AS1411 aptamer to facilitate the targeting and internalization of the probe into the MCF-7 cells. Under UV irradiation, the hairpin is cleaved, leading to the intracellular mRNA toehold-stimulated displacement of the FAM-functionalized strand resulting in a switched-on fluorescence signal upon the detection of the mRNA in a single cell. In addition, a nanoelectrode functionalized with a methylene blue (MB) redox-active photocleavable hairpin is inserted into the cytoplasm of a single MCF-7 cell. Photocleavage of the hairpin leads to the mRNA-mediated toehold displacement of the redox-active strand associated with the probe, leading to the depletion of the voltammetric response of the probe. The parallel optical and electrochemical detection of the mRNA at a single cell level is demonstrated

Engineering Biosensors with Dual Programmable Dynamic Ranges

Although extensively used in all fields of chemistry, molecular recognition still suffers from a significant limitation: host–guest binding displays a fixed, hyperbolic dose–response curve, which limits its usefulness in many applications. Here we take advantage of the high programmability of DNA chemistry and propose a universal strategy to engineer biorecognition-based sensors with dual programmable dynamic ranges. Using DNA aptamers as our model recognition element and electrochemistry as our readout signal, we first designed a dual signaling “signal-on” and “signal-off” adenosine triphosphate (ATP) sensor composed of a ferrocene-labeled ATP aptamer in complex to a complementary, electrode-bound, methylene-blue labeled DNA. Using this simple “dimeric” sensor, we show that we can easily (1) tune the dynamic range of this dual-signaling sensor through base mutations on the electrode-bound DNA, (2) extend the dynamic range of this sensor by 2 orders of magnitude by using a combination of electrode-bound strands with varying affinity for the aptamers, (3) create an ultrasensitive dual signaling sensor by employing a sequestration strategy in which a nonsignaling, high affinity “depletant” DNA aptamer is added to the sensor surface, and (4) engineer a sensor that simultaneously provides extended and ultrasensitive readouts. These strategies, applicable to a wide range of biosensors and chemical systems, should broaden the application of molecular recognition in various fields of chemistry

Erythrocyte Membrane-Camouflaged Aggregation-Induced Emission Nanoparticles for Fetal Intestinal Maturation Assessment

Assessment of fetal maturity is essential for timely termination of pregnancy, especially in pregnant women with pregnancy complications. However, there is a lack of methods to assess the maturity of fetal intestinal function. Here, we constructed erythrocyte membrane-camouflaged aggregation-induced emission (AIE) nanoparticles. Nanocore is formed using a hollow mesoporous silicon nanobox (HMSN) of different particle sizes loaded with AIE luminogens -PyTPA (P), which are then co-extruded with erythrocyte membranes (M) to construct M@HMSN@P. The 100 nm M@HMSN@P has a more effective cellular uptake efficiency in vitro and in vivo. Swallowing and intestinal function in fetal mice mature with the increase in gestational age. After intrauterine injection of M@HMSN@P, they were swallowed and absorbed by fetal mice, and their swallowed and absorbed amount was positively correlated with the gestational age with a correlation coefficient of 0.9625. Using the M@HMSN@P (fluorescence intensity) in fetal mice, the gestational age can be imputed, and the difference between this imputed gestational age and the actual gestational age is less than 1 day. Importantly, M@HMSN@P has no side effect on the health status of pregnant and fetal mice, showing good biocompatibility. In conclusion, we constructed M@HMSN@P nanoparticles with different particle sizes and confirmed that the smaller size M@HMSN@P has more efficient absorption efficiency and it can assess fetal intestinal maturity by the intensity of the fluorescence signal

Lab in a Tube: Ultrasensitive Detection of MicroRNAs at the Single-Cell Level and in Breast Cancer Patients Using Quadratic Isothermal Amplification

Through rational design of a functional molecular probe with high sequence specificity that takes advantage of sensitive isothermal amplification with simple operation, we developed a one-pot hairpin-mediated quadratic enzymatic amplification strategy for microRNA (miRNA) detection. Our method exhibits ultrahigh sensitivity toward miR-21 with detection limits of 10 fM at 37 °C and 1 aM at 4 °C, which corresponds to nine strands of miR-21 in a 15 μL sample, and it is capable of distinguishing among miRNA family members. More importantly, the proposed approach is also sensitive and selective when applied to crude extractions from MCF-7 and PC3 cell lines and even patient tissues from intraductal carcinoma and invasive ductal carcinoma of the breast

Assembly and Densification of Nanowire Arrays via Shrinkage

Chemically synthesized semiconductor nanowires (NWs) have demonstrated substantial promise for nanoelectronics, nanoenergy, and nanobiotechnology, but the lack of an effective and controllable assembly process has limited the wide adoption of NWs in these areas. Here we demonstrate a facile, robust, and controllable approach to assembling and densifying a parallel array of NWs using shrinkable shape memory polymers. Using thermal-induced shrinkage of polystyrene, we were able to successfully assemble and densify NW arrays up to close-packing and, furthermore, achieve tunable density (up to ∼300% amplification of density) by controlling the shrinkage process. We also demonstrate scalable assembly and densification of NWs on a 2.5 × 6 inch scale to explore the manufacturability of the shrink-induced assembly process. Finally, we demonstrate the successful transfer of the shrink-assembled NW arrays onto various 2-dimensional and 3-dimensional substrates without compromising the integrity of NW assembly and density