A simple and versatile CRISPR/Cas12a-based immunosensing platform: Towards attomolar level sensitivity for small protein diagnostics

Recent advances in CRISPR/Cas biosensing have led to impressive performance in sensitivity, specificity, and speed for nucleic acid detection. However, the remarkable advantages (such as universality, ultralow, attomolar detection limits) of CRISPR/Cas biosensing systems are limited in testing non-nucleic acid targets. Herein, by synthesizing a functional hybrid conjugate of antibody and single strand DNA oligonucleotide, we had successfully demonstrated the capability to integrate CRISPR/Cas12a-based signal amplification into different types of immunoassay schemes without the need for any additional recognition molecule or molecular synthesis during the detection process, thus providing a simple but generally applicable approach to improve the conventional immunoassays with attomolar sensitivity for small protein detections, referred as the CRISPR-based Universal Immunoassay Signal Enhancer (CRUISE). CRUISE is capable of being integrated into various immunoassays either through the primary antibody or the secondary antibody, with sensitivity down to 1 fg mL−1 (∼50 aM) and 6 logs of linear range for detecting cytokines, such as IFN-γ and EGFR, under 3–4 h. It has a 103 times higher sensitivity compared to a commercial IFN-γ ELISA kit, but uses the same experimental scheme. The same 1 fg mL−1 sensitivity along with 6 logs of linear range was realized for IFN-γ detection in human plasma samples. We are expecting that our CRUISE provides an alternative but simple, user-friendly and effective strategy for those who rely on the use of immunoassays, while struggling with the limits of their sensitivity or detection ranges

Probing Carrier Behavior at the Nanoscale in Gallium Nitride using Low Voltage Cathodoluminescence

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X-ray radiation-induced and targeted photodynamic therapy with folic acid-conjugated biodegradable nanoconstructs.

INTRODUCTION: The depth limitation of conventional photodynamic therapy (PDT) with visible electromagnetic radiation represents a challenge for the treatment of deep-seated tumors. MATERIALS AND METHODS: To overcome this issue, we developed an X-ray-induced PDT system where poly(lactide-co-glycolide) (PLGA) polymeric nanoparticles (NPs) incorporating a photosensitizer (PS), verteporfin (VP), were triggered by 6 MeV X-ray radiation to generate cytotoxic singlet oxygen. The X-ray radiation used in this study allows this system to breakthrough the PDT depth barrier due to excellent penetration of 6 MeV X-ray radiation through biological tissue. In addition, the conjugation of our NPs with folic acid moieties enables specific targeting of HCT116 cancer cells that overexpress the folate receptors. We carried out physiochemical characterization of PLGA NPs, such as size distribution, zeta potential, morphology and in vitro release of VP. Cellular uptake activity and cell-killing effect of these NPs were also evaluated. RESULTS AND DISCUSSION: Our results indicate that our nanoconstructs triggered by 6 MeV X-ray radiation yield enhanced PDT efficacy compared with the radiation alone. We attributed the X-ray-induced singlet oxygen generation from the PS, VP, to photoexcitation by Cherenkov radiation and/or reactive oxygen species generation facilitated by energetic secondary electrons produced in the tissue. CONCLUSION: The cytotoxic effect caused by VP offers the possibility of enhancing the radiation therapy commonly prescribed for the treatment of cancer by simultaneous PDT

Light-triggered liposomal cargo delivery platform incorporating photosensitizers and gold nanoparticles for enhanced singlet oxygen generation and increased cytotoxicity.

We developed light-triggered liposomes incorporating 3-5 nm hydrophobic gold nanoparticles and Rose Bengal (RB), a well-known photosensitizer used for photodynamic therapy. Singlet oxygen generated by these liposomes with 532 nm light illumination was characterized for varying the molar ratio of lipids and gold nanoparticles while keeping the amount of RB constant. Gold nanoparticles were found to enhance the singlet oxygen generation rate, with a maximum enhancement factor of 1.75 obtained for the molar ratio of hydrogenated soy l-α-phosphatidylcholine:1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(hexanoylamine):gold of 57:5:17 compared with liposomes loaded with RB alone. The experimental results could be explained by the local electric field enhancement caused by gold nanoparticles. We further assessed cellular cytotoxicity of gold-loaded liposomes by encapsulating an antitumor drug, doxorubicin (Dox); such Dox-loaded liposomes were applied to human colorectal cancer cells (HCT116) and exposed to light. Gold-loaded liposomes containing RB and Dox where Dox release was triggered by light were found to exhibit higher cytotoxicity compared with the liposomes loaded with RB and Dox alone. Our results indicate that gold-loaded liposomes incorporating photosensitizers may serve as improved agents in photodynamic therapy and chemotherapy

Increasing trans-cleavage catalytic efficiency of Cas12a and Cas13a with chemical enhancers: Application to amplified nucleic acid detection

The exceptional programable trans-cleavage ability of type V and VI CRISPR/Cas nucleases paved the way for ultrasensitive CRISPR/Cas based sensing of nucleic acid and alternative targets. However, the enhancement of the trans-cleavage activity of Cas effector with organic chemical agents has not been explored thus far. We report here chemically enhanced trans-cleavage activity of Cas12a and Cas13a nucleases which improves sensor performance in CRISPR/Cas biosensing. Improved trans-ssDNA cleavage of Cas12a and trans-ssRNA cleavage of Cas13a were demonstrated by using sulfhydryl reductants and non-ionic surfactants. DTT and PVA were demonstrated to be the most effective chemical enhancers in both cases. By using a fluorescence resonance energy transfer (FRET)-based intramolecular distance measurements, we identified the mechanism of this enhancement to be the conformation change of the ribonucleoprotein and quantified it to be major (about 50% increase of a relevant intramolecular distance). These chemical enhancers have been integrated into the established CRISPR/Cas biosensing protocols without additional modifications. For the detection of Helicobacter Pylori DNA and SARS-CoV-2 RNA, we found a decreased reaction time by 75–83% and 4–6-fold increased sensitivity. These results indicate that chemical enhancers provide a versatile and broadly applicable approach to break through the barriers of long reaction time and sensitivity in CRISPR/Cas sensors

X-ray induced photodynamic therapy (PDT) with a mitochondria-targeted liposome delivery system.

In this study, we constructed multifunctional liposomes with preferentially mitochondria-targeted feature and gold nanoparticles-assisted synergistic photodynamic therapy. We systemically investigated the in vitro X-ray triggered PDT effect of these liposomes on HCT 116 cells including the levels of singlet oxygen, mitochondrial membrane potential, cell apoptosis/necrosis and the expression of apoptosis-related proteins. The results corroborated that synchronous action of PDT and X-ray radiation enhance the generation of cytotoxic reactive oxygen species produced from the engineered liposomes, causing mitochondrial dysfunction and increasing the levels of apoptosis

X-ray induced singlet oxygen generation by nanoparticle-photosensitizer conjugates for photodynamic therapy: determination of singlet oxygen quantum yield.

Singlet oxygen is a primary cytotoxic agent in photodynamic therapy. We show that CeF3 nanoparticles, pure as well as conjugated through electrostatic interaction with the photosensitizer verteporfin, are able to generate singlet oxygen as a result of UV light and 8 keV X-ray irradiation. The X-ray stimulated singlet oxygen quantum yield was determined to be 0.79 ± 0.05 for the conjugate with 31 verteporfin molecules per CeF3 nanoparticle, the highest conjugation level used. From this result we estimate the singlet oxygen dose generated from CeF3-verteporfin conjugates for a therapeutic dose of 60 Gy of ionizing radiation at energies of 6 MeV and 30 keV to be (1.2 ± 0.7) × 10(8) and (2.0 ± 0.1) × 10(9) singlet oxygen molecules per cell, respectively. These are comparable with cytotoxic doses of 5 × 10(7)-2 × 10(9) singlet oxygen molecules per cell reported in the literature for photodynamic therapy using light activation. We confirmed that the CeF3-VP conjugates enhanced cell killing with 6 MeV radiation. This work confirms the feasibility of using X- or γ- ray activated nanoparticle-photosensitizer conjugates, either to supplement the radiation treatment of cancer, or as an independent treatment modality

Nanoscale plasmonic resonators with high Purcell factor: Spontaneous and stimulated emission

Plasmonic nanoparticles with silver cores and silica shells containing Eu fluorophores near the surface have been produced by wet chemistry method and their spontaneous emission properties characterized. Fluorescence amplification and decreased lifetime is interpreted within the Purcell framework which highlights the role of surface plasmon polariton modes of the nanoparticle. These behave as energy-storing resonators, with values of the Q factor between 50 and 170 at the fluorophore wavelength of 615 nm, and very small mode volumes, in the order of 104 nm3, producing high Purcell factors of over 4000. Comparison of experiment with theoretical calculations by using the Mie theory shows that the values of cavity Q factors are moderated by the nonradiative rate of fluorophore molecules close to metal. The criteria for laser action in such composite nanoparticles are also presented, including lasing frequencies and threshold gain. © 2011 SPIE

Formation and dissociation of hydrogen-related defect centers in Mg-doped GaN

Moderately and heavily Mg-doped GaN were studied by a combination of post-growth annealing processes and electron beam irradiation techniques during cathodoluminescence (CL) to elucidate the chemical origin of the recombination centers responsible for the main optical emission lines. The shallow donor at 20-30 meV below the conduction band, which is involved in the donor-acceptor-pair (DAP) emission at 3.27 eV, was attributed to a hydrogen-related center, presumably a (VN-H) complex. Due to the small dissociation energy (<2 eV) of the (VNH) complex, this emission line was strongly reduced by low-energy electron irradiation. CL investigations of the DAP at a similar energetic position in Si-doped (n-type) GaN indicated that this emission line is of different chemical origin than the 3.27 eV DAP in Mg-doped GaN. A slightly deeper DAP emission centered at 3.14 eV was observed following low-energy electron irradiation, indicating the appearance of an additional donor level with a binding energy of 100-200 meV, which was tentatively attributed to a VN-related center. The blue band (2.8-3.0 eV) in heavily Mg-doped GaN was found to consist of at least two different deep donor levels at 350±30 meV and 440±40 meV. The donor level at 350±30 meV was strongly affected by electron irradiation and attributed to a H-related defect

Controlled gene and drug release from a liposomal delivery platform triggered by X-ray radiation.

Liposomes have been well established as an effective drug delivery system, due to simplicity of their preparation and unique characteristics. However conventional liposomes are unsuitable for the on-demand content release, which limits their therapeutic utility. Here we report X-ray-triggerable liposomes incorporating gold nanoparticles and photosensitizer verteporfin. The 6 MeV X-ray radiation induces verteporfin to produce singlet oxygen, which destabilises the liposomal membrane and causes the release of cargos from the liposomal cavity. This triggering strategy is demonstrated by the efficiency of gene silencing in vitro and increased effectiveness of chemotherapy in vivo. Our work indicates the feasibility of a combinatorial treatment and possible synergistic effects in the course of standard radiotherapy combined with chemotherapy delivered via X-ray-triggered liposomes. Importantly, our X-ray-mediated liposome release strategy offers prospects for deep tissue photodynamic therapy, by removing its depth limitation