Media 1: In vivo fast variable focus photoacoustic microscopy using an electrically tunable lens

Originally published in Optics Express on 25 August 2014 (oe-22-17-20130

Linear Ru(bpy)₃²⁺–Polymer as a Universal Probe for Sensitive Detection of Biomarkers with Controllable Electrochemiluminescence Signal-Amplifying Ratio

Electrochemiluminescence (ECL) has been engineered to perform various tasks in the area of immunoassays and molecular diagnosis. However, there is still substantial potential for developments of ECL assay with high efficiency to achieve trace analysis. Herein, we demonstrate a polymer-amplified ECL assay via construction of linear Ru­(bpy)₃²⁺–polymer. This new polymer material compensates for the relatively low ECL intensity from single ECL luminophore and realizes a stable and controllable labeling process. The polymer-amplified ECL assay achieved a remarkable sensitivity of 100 amol. The wide-ranging applications of the polymer-amplified ECL assay for Hepatitis B virus, carcinoembryonic antigen, 16sRNA, and thrombin also demonstrate its superiority. Hence, the polymer-amplified ECL assay possesses the potential to create a new paradigm in amplified ECL assays that could provide outstanding performance for biomedical analysis

<i>In Vitro</i> Evaluation of CRISPR/Cas9 Function by an Electrochemiluminescent Assay

The CRISPR/Cas9 system is a revolutionary genome-editing tool that enables targeted and efficient gene knockouts. However, the off-target effects and loci-dependent enzyme activity limit its uses on the field of research and treatment. In this study, we designed a convenient and sensitive <i>in vitro</i> test method, which was based on electrochemiluminescence (ECL) technology for evaluating cleavage activity of the CRISPR/Cas9 system. It was find that Cas9 can tolerate some common genetic modifications to its target DNA. It was also find that target DNA/sgRNA with single-base mismatch and UV damages of target DNA resulted in significantly reduction of Cas9 cleavage efficiency. Comparing with traditional method, the proposed method reduced the evaluation time from weeks to 2 h. Therefore, our study provides a versatile <i>in vitro</i> method for a priori analysis of CRISPR/Cas9 system and highlights the potential to guide <i>in vivo</i> genome editing

Clustered Regularly Interspaced Short Palindromic Repeats/Cas9 Triggered Isothermal Amplification for Site-Specific Nucleic Acid Detection

A novel CRISPR/Cas9 triggered isothermal exponential amplification reaction (CAS-EXPAR) strategy based on CRISPR/Cas9 cleavage and nicking endonuclease (NEase) mediated nucleic acids amplification was developed for rapid and site-specific nucleic acid detection. CAS-EXPAR was primed by the target DNA fragment produced by cleavage of CRISPR/Cas9, and the amplification reaction performed cyclically to generate a large number of DNA replicates which were detected using a real-time fluorescence monitoring method. This strategy that combines the advantages of CRISPR/Cas9 and exponential amplification showed high specificity as well as rapid amplification kinetics. Unlike conventional nucleic acids amplification reactions, CAS-EXPAR does not require exogenous primers, which often cause target-independent amplification. Instead, primers are first generated by Cas9/sgRNA directed site-specific cleavage of target and accumulated during the reaction. It was demonstrated this strategy gave a detection limit of 0.82 amol and showed excellent specificity in discriminating single-base mismatch. Moreover, the applicability of this method to detect DNA methylation and <i>L. monocytogenes</i> total RNA was also verified. Therefore, CAS-EXPAR may provide a new paradigm for efficient nucleic acid amplification and hold the potential for molecular diagnostic applications

Media 2: In vivo fast variable focus photoacoustic microscopy using an electrically tunable lens

Originally published in Optics Express on 25 August 2014 (oe-22-17-20130

<i>In Vivo</i> Imaging-Guided Photothermal/Photoacoustic Synergistic Therapy with Bioorthogonal Metabolic Glycoengineering-Activated Tumor Targeting Nanoparticles

Developing multifunctional phototheranostics with nanoplatforms offers promising potential for effective eradication of malignant solid tumors. In this study, we develop a multifunctional phototheranostic by combining photothermal therapy (PTT) and photoacoustic therapy (PAT) based on a tumor-targeting nanoagent (DBCO-ZnPc-LP). The nanoagent DBCO-ZnPc-LP was facilely prepared by self-assembling of a single lipophilic near-infrared (NIR) dye zinc­(II)-phthalocyanine (ZnPc) with a lipid-poly­(ethylene glycol) (LP) and following modified further with dibenzyl cyclootyne (DBCO) for introducing the two-step chemical tumor-targeting strategy based on metabolic glycoengineering and click chemistry. The as-prepared DBCO-ZnPc-LP could not only convert NIR light into heat for effective thermal ablation but also induce a thermal-enhanced ultrasound shockwave boost to trigger substantially localized mechanical damage, achieving synergistic antitumor effect both <i>in vitro</i> and <i>in vivo</i>. Moreover, DBCO-ZnPc-LP can be efficiently delivered into tumor cells and solid tumors after being injected intravenously <i>via</i> the two-step tumor-targeting strategy. By integrating the targeting strategy, photoacoustic imaging, and the synergistic interaction between PTT and PAT, a solid tumor could be accurately positioned and thoroughly eradicated <i>in vivo</i>. Therefore, this multifunctional phototheranostic is believed to play an important role in future oncotherapy by the enhanced synergistic effect of PTT and PAT under the guidance of photoacoustic imaging

Paper-Based Bipolar Electrode Electrochemiluminescence Switch for Label-Free and Sensitive Genetic Detection of Pathogenic Bacteria

Genetic analysis is of great importance for the detection of pathogenic bacteria. Bacterial identification must become simpler, less expensive, and more rapid than the traditional methods. In this study, a low-cost, label-free, and wireless paper-based bipolar electrode electrochemiluminescence (pBPE-ECL) analysis system was constructed for the rapid and sensitive genetic detection of pathogenic bacteria. Wax-screen printing was used to form hydrophilic channels on filter paper, and a carbon ink-based bipolar electrode and driving electrodes were screen-printed into the channels. The “light-switch” molecule [Ru­(phen)₂dppz]²⁺ (phen = 1,10-phenanthroline; dppz = dipyridophenazine) was used to intercalate into the base pairs of the double-stranded DNA PCR amplification products, and the complexs were then applied to the paper-based bipolar electrode to perform the ECL assays; the ECL of [Ru­(phen)₂dppz]²⁺ is quenched in aqueous solution, but this molecule displays intense ECL when intercalated into double-stranded DNA. Under optimized experimental conditions, as little as 10 copies/μL of the genomic DNA of <i>Listeria monocytogenes</i> could be detected. Additionally, the system could also specifically distinguish <i>Listeria monocytogenes</i> from <i>Salmonella</i>, <i>Escherichia coli</i> O157:H7, and <i>Staphylococcus aureus</i>. This label-free, simple, and rapid method has potential in point-of-care applications for pathogen detection

Pyropheophorbide A and c(RGDyK) Comodified Chitosan-Wrapped Upconversion Nanoparticle for Targeted Near-Infrared Photodynamic Therapy

Near-infrared (NIR)-to-visible upconversion nanoparticle (UCNP) has shown promising prospects in photodynamic therapy (PDT) as a drug carrier or energy donor. In this work, a photosensitizer pyropheophorbide a (Ppa) and RGD peptide c­(RGDyK) comodified chitosan-wrapped NaYF₄:Yb/Er upconversion nanoparticle UCNP-Ppa-RGD was developed for targeted near-infrared photodynamic therapy. The properties of UCNP-Ppa-RGD, such as morphology, stability, optical spectroscopy and singlet oxygen generation efficiency, were investigated. The results show that covalently linked pyropheophorbide a molecule not only is stable but also retains its spectroscopic and functional properties. In vitro studies confirm a stronger targeting specificity of UCNP-Ppa-RGD to integrin α_vβ₃-positive U87-MG cells compared with that in the corresponding negative group. The photosensitizer-attached nanostructure exhibited low dark toxicity and high phototoxicity against cancer cells upon 980 nm laser irradiation at an appropriate dosage. These results represent the first demonstration of a highly stable and efficient photosensitizer modified upconversion nanostructure for targeted near-infrared photodynamic therapy of cancer cells. The novel UCNP-Ppa-RGD nanoparticle may provide a powerful alternative for near-infrared photodynamic therapy with an improved tumor targeting specificity

Ultrasensitive Detection of MicroRNA in Tumor Cells and Tissues via Continuous Assembly of DNA Probe

Nucleic acids have been engineered to participate in a wide variety of tasks. Among them, the enzyme-free amplification modes, enzyme-free DNA circuits (EFDCs), and hybridization chain reactions (HCRs) have been widely applied in a series of studies of bioanalysis. We demonstrated here an ultrasensitive hairpin probe-based circulation for continuous assemble of DNA probe. This strategy improved the analyte stability-dependent amplification efficiency of EFDC and signal enhancement without being limited by the analyte’s initial concentration, and it was used to produce a novel microRNA (miRNA) trace analysis assay with ultrasensitive amplification properties. Through the detection of standard miRNA substances, 1 amol-level sensitivity and satisfactory specificity were achieved. Compared with EFDCs and HCRs, the sensitivity of ultrasensitive hairpin probe-based circulation was higher by 3 or 4 orders of magnitude. Furthermore, the excellent performance of this platform was also demonstrated in the detection of miRNAs in tumor cells. The sensitivities for the detection of miRNAs in HepG2, A549 and MCF-7 tumor cells were 10, 10, and 100 cells, respectively. In addition, a high detection rate of 83% was achieved for tumor tissues. Thus, this ultrasensitive hairpin probe-based circulation possesses the potential to be a technological innovation in the field of tumor diagnosis

Simultaneous Detection of Antibiotic Resistance Genes on Paper-Based Chip Using [Ru(phen)₂dppz]²⁺ Turn-on Fluorescence Probe

Antibiotic resistance, the ability of some bacteria to resist antibiotic drugs, has been a major global health burden due to the extensive use of antibiotic agents. Antibiotic resistance is encoded via particular genes; hence the specific detection of these genes is necessary for diagnosis and treatment of antibiotic resistant cases. Conventional methods for monitoring antibiotic resistance genes require the sample to be transported to a central laboratory for tedious and sophisticated tests, which is grueling and time-consuming. We developed a paper-based chip, integrated with loop-mediated isothermal amplification (LAMP) and the “light switch” molecule [Ru­(phen)₂dppz]²⁺, to conduct turn-on fluorescent detection of antibiotic resistance genes. In this assay, the amplification reagents can be embedded into test spots of the chip in advance, thus simplifying the detection procedure. [Ru­(phen)₂dppz]²⁺ was applied to intercalate into amplicons for product analysis, enabling this assay to be operated in a wash-free format. The paper-based detection device exhibited a limit of detection (LOD) as few as 100 copies for antibiotic resistance genes. Meanwhile, it could detect antibiotic resistance genes from various bacteria. Noticeably, the approach can be applied to other genes besides antibiotic resistance genes by simply changing the LAMP primers. Therefore, this paper-based chip has the potential for point-of-care (POC) applications to detect various gene samples, especially in resource-limited conditions