DNA Origami Plasmonic Nanoantenna for Programmable Biosensing of Multiple Cytokines in Cancer Immunotherapy.

Herein, we report a DNA origami plasmonic nanoantenna for the programmable surface-enhanced Raman scattering (SERS) detection of cytokine release syndrome (CRS)-associated cytokines (e.g., tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ)) in cancer immunotherapy. Typically, the nanoantenna was made of self-assembled DNA origami nanotubes (diameter: ∼19 nm; length: ∼90 nm) attached to a silver nanoparticle-modified silicon wafer (AgNP/Si). Each DNA origami nanotube contains one miniature gold nanorod (AuNR) inside (e.g., length: ∼35 nm; width: ∼7 nm). Intriguingly, TNF-α and IFN-γ logically regulate the opening of the nanotubes and the dissociation of the AuNRs from the origami structure upon binding to their corresponding aptamers. On this basis, we constructed a complete set of Boolean logic gates that read cytokine molecules as inputs and return changes in Raman signals as outputs. Significantly, we demonstrated that the presented system enables the quantification of TNF-α and IFN-γ in the serum of tumor-bearing mice receiving different types of immunotherapies (e.g., PD1/PD-L1 complex inhibitors and STING agonists). The sensing results are consistent with those of the ELISA. This strategy fills a gap in the use of DNA origami for the detection of multiple cytokines in real systems.

CRISPR Cas12a-Powered Silicon Surface-Enhanced Raman Spectroscopy Ratiometric Chip for Sensitive and Reliable Quantification.

Sensitive and reliable clustered regularly interspaced short palindromic repeats (CRISPR) quantification without preamplification of the sample remains a challenge. Herein, we report a CRISPR Cas12a-powered silicon surface-enhanced Raman spectroscopy (SERS) ratiometric chip for sensitive and reliable quantification. As a proof-of-concept application, we select the platelet-derived growth factor-BB (PDGF-BB) as the target. We first develop a microfluidic synthetic strategy to prepare homogeneous silicon SERS substrates, in which uniform silver nanoparticles (AgNPs) are in situ grown on a silicon wafer (AgNPs@Si) by microfluidic galvanic deposition reactions. Next, one 5'-SH-3'-ROX-labeled single-stranded DNA (ssDNA) is modified on AgNPs via Ag-S bonds. In our design, such ssDNA has two fragments: one fragment hybridizes to its complementary DNA (5'-Cy3-labeled ssDNA) to form double-stranded DNA (dsDNA) and the other fragment labeled with 6'-carboxy-X-rhodmine (ROX) extends out as a substrate for Cas12a. The cleavage of the ROX-tagged fragment by Cas12a is controlled by the presence or not of PDGF-BB. Meanwhile, Cy3 molecules serving as internal standard molecules still stay at the end of the rigid dsDNA, and their signals remain constant. Thereby, the ratio of ROX signal intensity to Cy3 intensity can be employed for the reliable quantification of PDGF-BB concentration. The developed chip features an ultrahigh sensitivity (e.g., the limit of detection is as low as 3.2 pM, approximately 50 times more sensitive than the fluorescence counterpart) and good reproducibility (e.g., the relative standard deviation is less than 5%) in the detection of PDGF-BB.

NIR-II Photoacoustic-Active DNA Origami Nanoantenna for Early Diagnosis and Smart Therapy of Acute Kidney Injury.

Herein, we designed and synthesized a novel microRNA (miR)-responsive nanoantenna capable of early diagnosis and smart treatment of acute kidney injury (AKI). The nanoantenna was made of two miniature gold nanorods (AuNRs) (e.g., length: ∼48 nm; width: ∼9 nm) linked together by a rectangular DNA origami nanostructure (rDONs) scaffold (e.g., length: ∼90 nm; width: ∼60 nm) (rDONs@AuNR dimer). The surface plasmon resonance peak of the constructed nanoantenna is located within the NIR-II window (e.g., ∼1060 nm), thus guaranteeing photoacoustic (PA) imaging of the nanoantenna in deep tissues. Intriguingly, the nanoantenna displayed exclusive kidney retention in both healthy mice and ischemia reperfusion-induced AKI mice by leveraging the kidney-targeting ability of rDONs. Distinguished from the stable signals in the healthy mice, the PA signals of the nanoantenna would turn down in the AKI mice due to the AuNR detached from rDONs upon interaction with miR-21, which were up-expressed in AKI mice. The limit of detection toward miR-21 was down to 2.8 nM, enabling diagnosis of AKI as early as 10 min post-treatment with ischemia reperfusion, around 2 orders of magnitude earlier than most established probes. Moreover, the naked rDON scaffold generated by AKI could capture more reactive oxygen species (e.g., 1.5-fold more than rDONs@AuNR dimer), alleviating ischemic AKI. This strategy provided a new avenue for early diagnosis and smart treatment of AKI.

In Situ Monitoring of Dynamic Photocatalysis of Metal-Organic Frameworks by Three-Dimensional Shell-Isolated Nanoparticle-Enhanced Raman Spectroscopy.

Metal-organic frameworks (MOFs) are promising as novel disinfectants due to the reactive oxygen species (ROS) produced in their photocatalytic processes. The optimal MOF is screened as the best disinfectant, representing high-efficacy production of ROS under photocatalytic conditions. However, current methods to screen abundant MOFs for disinfectant application are generally semiquantitative or ex situ methods [such as electron paramagnetic resonance (EPR) measurements], so achieving a strategy that can quantitatively screen an optimal MOF in situ and is reliable is demanded. Herein, we developed a three-dimensional (3D) shell-isolated nanoparticle-enhanced Raman spectroscopy (SHINERS) platform to study the dynamic photocatalytic processes of various MOFs (e.g., ZIF-67, ZIF-8, and UIO-66) in situ. This platform comprises silica shell-isolated gold nanoparticles (AuNPs) modified on silicon nanowire arrays (SiNWArs). The MOF is then self-assembled on the 3D-SHINERS substrate. Using this platform, we recorded dynamic spectroscopic evidence of ROS formation by various MOFs under sunlight irradiation. By dynamic comparison, ZIF-67 has the most robust photocatalytic efficiency, ∼1.7-fold stronger than that of ZIF-8 and ∼42.6-fold stronger than that of UIO-66. As expected, ZIF-67 displays the best antibacterial ability, up to 99% in the agar plate assay. This work provides a versatile platform for dynamically monitoring photocatalytic performance and screening antibacterial MOFs.

Ultrasensitive discrimination of volatile organic compounds using a microfluidic silicon SERS artificial intelligence chip.

Current gaseous sensors hardly discriminate trace volatile organic compounds at the ppt level. Herein, we present an integrated platform for simultaneously enabling rapid preconcentration, reliable surface-enhanced Raman scattering, (SERS) detection and automatic identification of trace aldehydes at the ppt level. For rapid preconcentration, we demonstrate that the nozzle-like microfluidic concentrator allows the enrichment of rare gaseous analytes by five-fold in only 0.01 ms. The enriched gas is subsequently captured and detected by an integrated silicon-based SERS chip, which is made of zeolitic imidazolate framework-8 coated silver nanoparticles grown in situ on a silicon wafer. After SERS measurement, a fully connected deep neural network is built to extract faint features in the spectral dataset and discriminate volatile organic compound classes. We demonstrate that six kinds of gaseous aldehydes at 100 ppt could be detected and classified with an identification accuracy of ∼80.9% by using this platform.

In vivo bioluminescence imaging of natural bacteria within deep tissues via ATP-binding cassette sugar transporter.

Most existing bioluminescence imaging methods can only visualize the location of engineered bacteria in vivo, generally precluding the imaging of natural bacteria. Herein, we leverage bacteria-specific ATP-binding cassette sugar transporters to internalize luciferase and luciferin by hitchhiking them on the unique carbon source of bacteria. Typically, the synthesized bioluminescent probes are made of glucose polymer (GP), luciferase, Cy5 and ICG-modified silicon nanoparticles and their substrates are made of GP and D-luciferin-modified silicon nanoparticles. Compared with bacteria with mutations in transporters, which hardly internalize the probes in vitro (i.e., ~2% of uptake rate), various bacteria could robustly engulf the probes with a high uptake rate of around 50%. Notably, the developed strategy enables ex vivo bioluminescence imaging of human vitreous containing ten species of pathogens collected from patients with bacterial endophthalmitis. By using this platform, we further differentiate bacterial and non-bacterial nephritis and colitis in mice, while their chemiluminescent counterparts are unable to distinguish them.

Surface-enhanced Raman scattering holography chip for rapid, sensitive and multiplexed detection of human breast cancer-associated MicroRNAs in clinical samples.

MicroRNAs (miRNAs) are promising biomarkers for the early diagnosis of breast cancer. Yet, simultaneous achievement of rapid, sensitive and accurate detection of diverse miRNAs in clinical samples is still challenging due to the low abundance of miRNAs and the complex procedures of RNA extraction and separation. Herein, we develop an innovative three-dimensional (3D) surface-enhanced Raman scattering (SERS) holography sensing strategy for rapid, sensitive and multiplexed detection of human breast cancer-associated miRNAs. To establish a proof of concept, nine kinds of human breast cancer-associated miRNAs are isothermally amplified by Exonuclease (Exo) III enzyme, and the products could be spatially separated to corresponding sensing region on silicon SERS substrates. Each region has been modified with corresponding hairpin DNA probes, which are used to identify and quantify the miRNAs. Different DNA probes are labeled with different Raman reporters, which serve as "SERS tags" to incorporate spectroscopic information into computer-generated 3D SERS hologram within ~9 min. We demonstrate that 3D SERS holography chip not only achieves an ultrahigh sensitivity down to ~1 aM but also feature a high correlation with RT-qPCR in the detection of nine miRNAs in 30 clinical serum samples. This work provides a feasible tool to improve the diagnosis of breast cancer.