Single‐Cell Detection of mRNA Expression Using Nanofountain‐Probe Electroporated Molecular Beacons

New techniques for single‐cell analysis enable new discoveries in gene expression and systems biology. Time‐dependent measurements on individual cells are necessary, yet the common single‐cell analysis techniques used today require lysing the cell, suspending the cell, or long incubation times for transfection, thereby interfering with the ability to track an individual cell over time. Here a method for detecting mRNA expression in live single cells using molecular beacons that are transfected into single cells by means of nanofountain probe electroporation (NFP‐E) is presented. Molecular beacons are oligonucleotides that emit fluorescence upon binding to an mRNA target, rendering them useful for spatial and temporal studies of live cells. The NFP‐E is used to transfect a DNA‐based beacon that detects glyceraldehyde 3‐phosphate dehydrogenase and an RNA‐based beacon that detects a sequence cloned in the green fluorescence protein mRNA. It is shown that imaging analysis of transfection and mRNA detection can be performed within seconds after electroporation and without disturbing adhered cells. In addition, it is shown that time‐dependent detection of mRNA expression is feasible by transfecting the same single cell at different time points. This technique will be particularly useful for studies of cell differentiation, where several measurements of mRNA expression are required over time.     

Smart Magnetic Fluorescent Nanoparticle Imaging Probes to Monitor MicroRNAs

An imaging system that can be used to evaluate the expression levels of microRNAs during neuronal development can provide noninvasive information for investigating a variety of biological phenomena related to microRNAs (miRNAs, miRs). Herein, the development of a novel imaging platform to monitor intracellular miR124a during neuronal differentiation is reported using rhodamine‐coated cobalt ferrite magnetic fluorescent (MF) nanoparticles linked to a quenching molecular system containing an miR124a binding sequence (MF‐miR124a beacon). During neuronal differentiation, in vitro fluorescence signals of the MF‐miR124a beacon are significantly increased under conditions where miR124a is highly expressed, and dramatically return to the original quenched fluorescence after anti‐miR124a treatment. In vivo fluorescence images show enhanced fluorescence signals in mice with P19 cells within a poly‐L ‐lactic acid scaffold after induction of neuronal differentiation. In addition, magnetic resonance (MR) images provide in vivo tracking of cells containing the MF‐miR124a beacon. These studies represent the first step toward the use of nanotechnological imaging of mature miRNA, and this technique could be used for cellular tracking with a MR imaging system as well as for simultaneous monitoring of the miRNA expression pattern in vivo.     

肽类树状大分子及其生物医学应用

肽类树状大分子是近年来发展起来的一类新型生物医用高分子材料,它除了具有普通树枝状分子的特征如规整性、高度支化、表面呈现高密度功能团、尺度为纳米级、单一分子量等之外,同时还具有类似蛋白一样的球状结构、优良的生物相容性、水溶性、耐蛋白酶水解、生物降解等独特的性能。肽类树状大分子的上述特点,使它在生物医学应用中显示出诱人的前景。系统论述了肽类树状大分子的合成方法、以及在疾病诊断与治疗等生物医学领域中的应用。如作为MRI(磁共振成像)分子探针、诊断试剂以及基因治疗试剂等。将造影官能团偶联到肽类树枝状分子上,即得肽类树枝状MRI分子探针,该类MRI分子探针具有优良的生物相容性,纳米级尺寸结构。含氨基的肽类树枝状分子与基因(DNA)复合成纳米尺寸的粒子,可有效进入细胞,将DNA转移到目标部位,达到基因转染的目的。     

分子影像学研究的进展和前景

医学影像学历经百年,终于从解剖结构为成像基础的传统医学影像学发展到了建立在一细胞、分子结构和功能为基础的分子影像学时代,这代表了医学影像学的未来,将对现代和未来医学模式产生划时代的影响。本文扼要介绍近年来世界各国在分子影像学研究方面取得的丰硕成果和展望其在未来的光辉发展前景。     

Photoacoustic Probes for Molecular Detection: Recent Advances and Perspectives

Photoacoustic (PA) imaging (PAI) is a noninvasive and nonionizing biomedical imaging modality that combines the advantages of optical imaging and ultrasound imaging. Based on PAI, photoacoustic detection (PAD) is an emerging approach that is involved with the interaction between PA probes and analytes resulting in the changes of photoacoustic signals for molecular detection with rich contrast, high resolution, and deep tissue penetration. This Review focuses on the recent development of PA probes in PAD. The following contents will be discussed in detail: 1) the construction of PA probes; 2) the applications and mechanisms of PAD to different types of analytes, including microenvironments, small biomolecules, or metal ions; 3) the challenges and perspectives of PA probes in PAD.     

Targeting Peptide‐Based Probes for Molecular Imaging and Diagnosis

A series of novel peptide‐based molecular probes for different biomarkers is highlighted herein. These probes can provide targeted recognition with high affinity, high specificity, high penetration, and rapid excretion ability. These sensitive peptides can achieve rapid and specific detection when they are conjugated with imaging moieties or are formed into nanoprobes, which can be adapted for in vivo molecular imaging in targeted diagnosis and therapy.     

分子影像学基本内涵概述

分子影像学是一门新发展的医学影像学学科。分子影像学通过发展新的造影技术、系统、试剂及方法,探查疾病发展过程中细胞、亚细胞和分子水平的异常变化,在尚无解剖学改变前检出异常,从而为及早发现疾病、正确诊断和有效治疗提供更为丰富多样的医疗手段。本文扼要介绍分子影像学的基本概念、成像原理和实际应用。     

Photoacoustic Imaging: Contrast Agents and Their Biomedical Applications

Photoacoustic (PA) imaging as a fast‐developing imaging technique has great potential in biomedical and clinical applications. It is a noninvasive imaging modality that depends on the light‐absorption coefficient of the imaged tissue and the injected PA‐imaging contrast agents. Furthermore, PA imaging provides superb contrast, super spatial resolution, and high penetrability and sensitivity to tissue functional characteristics by detecting the acoustic wave to construct PA images. In recent years, a series of PA‐imaging contrast agents are developed to improve the PA‐imaging performance in biomedical applications. Here, recent progress of PA contrast agents and their biomedical applications are outlined. PA contrast agents are classified according to their components and function, and gold nanocrystals, gold‐nanocrystal assembly, transition‐metal chalcogenides/MXene‐based nanomaterials, carbon‐based nanomaterials, other inorganic imaging agents, small organic molecules, semiconducting polymer nanoparticles, and nonlinear PA‐imaging contrast agents are discussed. The applications of PA contrast agents as biosensors (in the sensing of metal ions, pH, enzymes, temperature, hypoxia, reactive oxygen species, and reactive nitrogen species) and in bioimaging (lymph nodes, vasculature, tumors, and brain tissue) are discussed in detail. Finally, an outlook on the future research and investigation of PA‐imaging contrast agents and their significance in biomedical research is presented.     

Nanoparticles for Optical Molecular Imaging of Atherosclerosis

Molecular imaging contributes to future personalized medicine dedicated to the treatment of cardiovascular disease, the leading cause of mortality in industrialized countries. Endoscope‐compatible optical imaging techniques would offer a stand‐alone alternative and high spatial resolution validation technique to clinically accepted imaging techniques in the (intravascular) assessment of vulnerable atherosclerotic lesions, which are predisposed to initiate acute clinical events. Efficient optical visualization of molecular epitopes specific for vulnerable atherosclerotic lesions requires targeting of high‐quality optical‐contrast‐enhancing particles. In this review, we provide an overview of both current optical nanoparticles and targeting ligands for optical molecular imaging of atherosclerotic lesions and speculate on their applicability in the clinical setting.

     

Multiplexed Molecular Imaging of Fresh Tissue Surfaces Enabled by Convection‐Enhanced Topical Staining with SERS‐Coded Nanoparticles

There is a need for intraoperative imaging technologies to guide breast‐conserving surgeries and to reduce the high rates of re‐excision for patients in which residual tumor is found at the surgical margins during postoperative pathology analyses. Feasibility studies have shown that utilizing topically applied surface‐enhanced Raman scattering (SERS) nanoparticles (NPs), in conjunction with the ratiometric imaging of targeted versus untargeted NPs, enables the rapid visualization of multiple cell‐surface biomarkers of cancer that are overexpressed at the surfaces of freshly excised breast tissues. In order to reliably and rapidly perform multiplexed Raman‐encoded molecular imaging of large numbers of biomarkers (with five or more NP flavors), an enhanced staining method has been developed in which tissue surfaces are cyclically dipped into an NP‐staining solution and subjected to high‐frequency mechanical vibration. This dipping and mechanical vibration (DMV) method promotes the convection of the SERS NPs at fresh tissue surfaces, which accelerates their binding to their respective biomarker targets. By utilizing a custom‐developed device for automated DMV staining, this study demonstrates the ability to simultaneously image four cell‐surface biomarkers of cancer at the surfaces of fresh human breast tissues with a mixture of five flavors of SERS NPs (four targeted and one untargeted control) topically applied for 5 min and imaged at a spatial resolution of 0.5 mm and a raster‐scanned imaging rate of >5 cm² min^?1.     

Advanced Photoacoustic Imaging Applications of Near‐Infrared Absorbing Organic Nanoparticles

Progress of nanotechnology in recent years has stimulated fast development of nanoparticles in biomedical research. Photoacoustic (PA) imaging as an emerging non‐invasive technique in molecular imaging has improved imaging depth relative to conventional optical imaging, demonstrating great potential in clinical applications. The convergence of nanotechnology and PA imaging has enabled a broad spectrum of new opportunities in fundamental biology and translation medicine. This review focuses on the recent advances of organic nanoparticles in PA imaging applications. Near‐infrared absorbing organic nanoparticles are classified and discussed according to their different imaging applications, which include tumor imaging, gastrointestinal imaging, sentinel lymph node imaging, disease microenvironment imaging and real‐time drug imaging. The chemistry and PA properties of organic nanoparticles are discussed in details to highlight their own merits, and their challenges and perspectives in PA imaging are also discussed.     

Ratiometric Photoacoustic Molecular Imaging for Methylmercury Detection in Living Subjects

Photoacoustic molecular imaging is an emerging and promising diagnostic tool for heavy metal ions detection. Methylmercury (MeHg⁺) is one of the most potent neurotoxins, which damages the brain and nervous system of human beings through fish consumption. The development of a selective and sensitive method for MeHg⁺ detection is highly desirable. In this Communication, we develope a chemoselective photoacoustic sensor (LP‐hCy7) composed of the liposome (LP) and MeHg⁺‐responsive near‐infrared (NIR) cyanine dye (hCy7) for MeHg⁺ detection within living subjects, such as zebrafish and mouse. The as‐prepared LP‐hCy7 nanoprobe displays unique dual‐shift NIR absorbance peaks and produces a normalized turn‐on response after the reaction of MeHg⁺ and hCy7 through a mercury‐promoted cyclization reaction. The absorbance intensities of LP‐hCy7 nanoprobe at 690 and 860 nm are decreased and increased, respectively. The ratiometric photoacoustic signal (PA860/PA690) is noticeably increased in the presence of MeHg⁺. These findings not only provide a ratiometric photoacoustic molecular imaging probe for the detection of metal ions in vivo, but also provides a tool for spectroscopic photoacoustic molecular imaging.     

Molecular Imaging Using Nanoparticle Quenchers of Cerenkov Luminescence

Cerenkov luminescence (CL) imaging is an emerging technique that collects the visible photons produced by radioisotopes. Here, molecular imaging strategies are investigated that switch the CL signal off. The noninvasive molecularly specific detection of cancer is demonstrated utilizing a combination of clinically approved agents, and their analogues. CL is modulated in vitro in a dose dependent manner using approved small molecules (Lymphazurin), as well as the clinically approved Feraheme and other preclinical superparamagnetic iron oxide nanoparticles (SPIO). To evaluate the quenching of CL in vivo, two strategies are pursued. [¹⁸F]‐FDG is imaged by PET and CL in tumors prior to and following accumulation of nanoparticles. Initially, non‐targeted particles are administered to mice bearing tumors in order to attenuate CL. For targeted imaging, a dual tumor model (expressing the human somatostatin receptor subtype‐2 (hSSTr2) and a control negative cell line) is used. Targeting hSSTr2 with octreotate‐conjugated SPIO, quenched CL enabling non‐invasive distinction between tumors' molecular expression profiles is demonstrated. In this work, the quenching of Cerenkov emissions is demonstrated in several proof of principle models using a combination of approved agents and nanoparticle platforms to provide disease relevant information including tumor vascularity and specific antigen expression.     

Rapid,High‐Throughput,and Direct Molecular Beacon Delivery to Human Cancer Cells Using a Nanowire‐Incorporated and Pneumatic Pressure‐Driven Microdevice

Tracking and monitoring the intracellular behavior of mRNA is of paramount importance for understanding real‐time gene expression in cell biology. To detect specific mRNA sequences, molecular beacons (MBs) have been widely employed as sensing probes. Although numerous strategies for MB delivery into the target cells have been reported, many issues such as the cytotoxicity of the carriers, dependence on the random probability of MB transfer, and critical cellular damage still need to be overcome. Herein, we have developed a nanowire‐incorporated and pneumatic pressure‐driven microdevice for rapid, high‐throughput, and direct MB delivery to human breast cancer MCF‐7 cells to monitor survivin mRNA expression. The proposed microdevice is composed of three layers: a pump‐associated glass manifold layer, a monolithic polydimethylsiloxane (PDMS) membrane, and a ZnO nanowire‐patterned microchannel layer. The MB is immobilized on the ZnO nanowires by disulfide bonding, and the glass manifold and PDMS membrane serve as a microvalve, so that the cellular attachment and detachment on the MB‐coated nanowire array can be manipulated. The combination of the nanowire‐mediated MB delivery and the microvalve function enable the transfer of MB into the cells in a controllable way with high cell viability and to detect survivin mRNA expression quantitatively after docetaxel treatment.     

生物荧光成像用分子与纳米探针

生物荧光成像近年来发展迅速,应用广泛。荧光探针是其中的核心技术之一。有机染料、半导体量子点和上转换稀土纳米粒子是适用于生物荧光成像的三类主要的化学荧光探针。简要评述了这三类荧光探针的发光机制、典型的设计发展策略、主要的合成制备方法、以及生物成像应用实例。各类探针都在不断地改进、完善自身,呈现出优势互补,共同发展的格局。     

基于量子点的分子灯塔探针的制备及其在DNA探针中的应用

根据荧光共振能量转移理论合成出一种新颖的分子灯塔探针.由于CdTe量子点（QD s）的荧光发射光谱与DABCYL的紫外-可见吸收光谱有很好的重叠性,所以此种探针采用CdTe量子点作为能量给体,DABCYL作为能量受体.通过水相法合成出直径为2.5 nm的CdTe量子点,并且在偶联剂1-乙基-3-（3-二甲基氨丙基）碳二亚氨盐酸盐（EDC）作用下,与5-′NH2-DNA-DABCYL连接得到了分子灯塔探针.实验发现探针的荧光强度相比CdTe-DNA有明显的下降,最大能量转移效率为68.3%,表明CdTe QD s和DABCYL之间发生了荧光共振能量转移.结果表明,此种探针体系对于互补DNA及其变种有着很好的特异性,且其检测极限为5.170×10^-9mol/L.