Fluorescence Readout Research Articles

CRISPR-Cas12a Biosensor Array for Ultrasensitive Detection of Unamplified DNA with Single-Nucleotide Polymorphic Discrimination

Quantitative polymerase chain reaction as a powerful tool for DNA detection has been pivotal to a vast range of applications, including disease screening, food safety assessment, environmental monitoring, and many others. However, the essential target amplification step in combination with fluorescence readout poses a significant challenge to rapid and streamlined analysis. The discovery and engineering of the clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) technology have recently paved the way for a novel approach to nucleic acid detection, but the majority of current CRISPR-mediated DNA detection platforms are limited by insufficient sensitivity and still require target preamplification. Herein, we report a CRISPR-Cas12a-mediated graphene field-effect transistor (gFET) array, named CRISPR Cas12a-gFET, for amplification-free, ultrasensitive, and reliable detection of both single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA) targets. CRISPR Cas12a-gFET leverages the multiturnover trans-cleavage activity of CRISPR Cas12a for intrinsic signal amplification and ultrasensitivity of gFET. As demonstrated, CRISPR Cas12a-gFET achieves a limit of detection of 1 aM for the ssDNA human papillomavirus 16 synthetic target and 10 aM for the dsDNA Escherichia coli plasmid target without target preamplification. In addition, an array of 48 sensors on a single 1.5 cm × 1.5 cm chip is employed to improve data reliability. Finally, Cas12a-gFET demonstrates the capability to discriminate single-nucleotide polymorphisms. Together, the CRISPR Cas12a-gFET biosensor array provides a detection tool for amplification-free, ultrasensitive, reliable, and highly specific DNA detections.

Abstract 6372: In situ detection of various activated immune checkpoints via next-generation proximity ligation assays in tumor tissue

Abstract Immune checkpoints (ICs), which are inhibitory signaling pathways that can down-modulate the immune responses of T cells, are pivotal in peripheral tissues and for maintaining immune self-tolerance. Among the many molecularly defined IC proteins, some of the most studied ones are CTLA-4 and its binding partners important for early T-cell co-inhibition, along with the PD-1/PD-L1 axis proteins that carry out late co-inhibitory signals. Notably, in addition to antigen presenting and other immune cells, many tumors also express CTLA-4 and PD-L1, which facilitate tumor evasion from the immune system. The inhibition of these IC proteins has revolutionized the field of cancer therapy, but its efficiency has been limited to a poorly defined subset of patients. Patient outcomes are likely to have a stronger correlation to high levels of PD-1/PD-L1 interaction and other hallmarks of pathway activation, such as PD-1 phosphorylation and recruitment of SHP-2, than to the overexpression of a single IC protein alone. To improve the predictive value of tissue staining, we created an immuno-oncology line of ultrasensitive kits based on proximity ligation, which detect the activation of multiple ICs. Naveni™ PD1/PD-L1 is a tool for direct visualization of the interaction between PD-1 and PD-L1, and has been verified in various FFPE tumor tissues. Naveni™ pY PD1 sensitively detects PD1 phosphorylation, which is the first step in the PD-1/PD-L1 inhibitory pathway activation. To study the subsequent recruitment of SHP-2 to the activated PD-1 receptor, which is crucial in immune cells but dispensable when the pathway is activated between two tumor cells, one can use the upcoming Naveni™ PD1/SHP-2 kit. Furthermore, we are developing assays to observe the competing interactions of CTLA-4 and CD28 with their binding partners CD80 and CD86, the interplay of which is pivotal for the initiation of early co-inhibition. All kits can be used on consecutive tissue sections to obtain a comprehensive picture of tumor IC pathway activation, which may not always correspond to CTLA-4, PD-1 or PD-L1 overexpression. While these kits have a chromogenic readout applicable to brightfield microscopy, we also have preliminary data on a PD-1/PD-L1 assay with a fluorescent readout thanks to which it can also be multiplexed. The multiplex feature generates an immune profile which may help improve immunotherapeutic strategies. The Naveni™ assays can be applied in basic research to elucidate the interplay of IC axes and downstream molecules, in pre-clinical and clinical research to compare stainings with the existing IHC assays and evaluate the potential prognostic value of interaction detection, and in pharma, aiding the development of new drugs or bispecific antibodies. Citation Format: Desirée Edén, Ka I Au Ieong, Naomi Cook, Doroteya Raykova, Agata Zieba Wicher. In situ detection of various activated immune checkpoints via next-generation proximity ligation assays in tumor tissue. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6372.

Abstract 2021: Naveni TriFlex cell: An emerging method for simultaneous detection of free proteins and their interactions

Abstract Determining the levels of protein-protein interactions (PPI) is essential for the understanding of signal transduction, regulation of gene expression and mutation effects, infection mechanisms, identification of diagnostic markers, etc. Taking the already challenging task of PPI identification a step further, the next aim is to determine the levels of non-interacting proteins in parallel. The ability to concurrently monitor free and interacting proteins opens the door to studying their functional states and interplay, thus gaining deeper insight from a single staining experiment. Additionally, to accomplish this in situ, retaining the structural integrity of the cell, helps understand spatiotemporal communication between proteins in their native environment. To this end, we designed Naveni TriFlex Cell – a highly sensitive and specific proximity-based technology, relying on two user-determined primary antibodies against the targets of interest, and on proprietary TriFlex Navenibodies, which are antibody-based proximity reagents. TriFlex Cell detects total protein A (i.e., both free and in complex with B), total protein B, and the AB interaction. The detected A, B and AB signals are amplified and generate fluorescent readout in three channels corresponding to each protein pool. As proof of principle, we stained MCF7 cells for E-cadherin and β-catenin, which are known to interact in the adherens junctions. We observed highly abundant interactions at the cell membrane, some complexes and free proteins in the cytoplasm, and very low background in controls where either or both primary antibodies were omitted. These results attested to the high sensitivity and specificity of our method. Next, we explored the expression and interaction of Histone H3 and Lamin B, a nuclear envelope protein. Despite the compact structure of the nucleus, we were able to distinguish individual signals and abundant interactions, with free Lamin B particularly enriched in the nuclear membrane. In dividing cells, we observed diffuse Lamin signals in line with nuclear envelope breakdown, and more densely packed Histone H3 associated with chromatin. In contrast to non-mitotic cells, interactions were few, but increasing with the advance from metaphase to telophase. Finally, we stained against GM130, a Golgi complex protein, and COX1, a mitochondrial marker, as a biological non-interaction control experiment. TriFlex Cell successfully detected the individual proteins in their respective subcellular locations, and no complexes. In summary, our data demonstrate that Naveni TriFlex Cell is a sensitive and specific method that visualizes low and high abundant targets in various cell compartments and adds value by uncovering protein interplay, such as complex formation/dissolution under dynamic biological conditions. Citation Format: Axel Klaesson, Doroteya Raykova, Agata Zieba Wicher. Naveni TriFlex cell: An emerging method for simultaneous detection of free proteins and their interactions [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2021.

Abstract 3775: Multi-omic spatial analysis with simultaneous detection of small RNAs, mRNAs and proteins using the novel RNAscope™ Plus technology

Abstract Regulatory RNA molecules such as microRNAs (miRNA) and long non-coding RNAs play critical roles in regulating translation of mRNA to protein. Several miRNAs have been implicated in disease initiation and progression, especially in cancer. The regulatory mechanisms of miRNAs and other small RNAs such as antisense oligos (ASOs) and silencing RNAs (siRNA) have been exploited to develop oligonucleotide therapies for 1. undruggable targets, 2. achieving longer-term effects 3. rapid production of therapeutics and 4. lower drug development costs. These therapeutics have the promise to treat debilitating neurodegenerative diseases, rare and inherited disorders. RNA therapies are typically delivered as a part of nanoparticles or a viral vector. Regardless of the delivery mechanism, methods to understand the biodistribution of the vector, transgene expression and cell type identification in a spatial context is crucial to study the safety and efficacy of these therapies. Built on the flagship RNAscope technology, the RNAscope Plus assay can detect 1 small RNA and 3 mRNA targets using TSA-based fluorescent readouts and is compatible with the new fluorescent Vivid™ dyes. Fixed, fresh frozen samples and formalin fixed paraffin embedded (FFPE) tissues are supported by manual and automated workflows using the Leica Bond Rx system. We have leveraged this technology to investigate spatial expression profile of miRNA and associated RNA targets across different tissue types and applications. We can also combine protein detection using a target antibody to visualize cell-type specific markers. This assay was used to demonstrate expression of miRNAs and target genes implicated in tumor initiation, progression, and angiogenesis. Expression of miR-205, associated tumor target genes such as PanCK, PTEN and tumor suppressor TP53 was visualized in head and neck cancer tumors. Downregulation of tumor-suppressor, TP53 resulted in upregulation of miR-205 which downregulates PTEN expression. Pan-CK stained the tumor region in the tissue. Similarly, miR-155 expression was observed in niche areas within breast cancer, head and neck cancer and cervical cancer tumors. Expression of miR-155 demonstrated correlation with high VEGF expression suggesting its role in angiogenesis. This novel platform will enable researchers to visualize regulatory RNA simultaneously with target RNAs, cell-type and morphology markers in intact cells/tissues with single cell resolution. This technology can provide meaningful insights into disease pathology driven by miRNAs as well as assess biodistribution and efficacy of oligonucleotide therapeutics. Citation Format: Anushka Dikshit, Sayantani Basak, Sonali Deshpande, Manvir Sambhi, Li-Chong Wang, Maithreyan Srinivasan. Multi-omic spatial analysis with simultaneous detection of small RNAs, mRNAs and proteins using the novel RNAscope™ Plus technology. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3775.

Photoisomerization of Heptamethine Cyanine Dyes Results in Red-Emissive Species: Implications for Near-IR, Single-Molecule, and Super-Resolution Fluorescence Spectroscopy and Imaging.

Photoisomerization kinetics of the near-infrared (NIR) fluorophore Sulfo-Cyanine7 (SCy7) was studied by a combination of fluorescence correlation spectroscopy (FCS) and transient state (TRAST) excitation modulation spectroscopy. A photoisomerized state with redshifted emission was identified, with kinetics consistent with a three-state photoisomerization model. Combining TRAST excitation modulation with spectrofluorimetry (spectral-TRAST) further confirmed an excitation-induced redshift in the emission spectrum of SCy7. We show how this red-emissive photoisomerized state contributes to the blinking kinetics in different emission bands of NIR cyanine dyes, and how it can influence single-molecule, super-resolution, as well as Förster resonance energy transfer (FRET) and multicolor readouts. Since this state can also be populated at moderate excitation intensities, it can also more broadly influence fluorescence readouts, also readouts not relying on high excitation conditions. However, this additional red-emissive state and its photodynamics, as identified and characterized in this work, can also be used as a strategy to push the emission of NIR cyanine dyes further into the NIR and to enhance photosensitization of nanoparticles with absorption spectra further into the NIR. Finally, we show that the photoisomerization kinetics of SCy7 and the formation of its redshifted photoisomer depend strongly on local environmental conditions, such as viscosity, polarity, and steric constraints, which suggests the use of SCy7 and other NIR cyanine dyes as environmental sensors. Such environmental information can be monitored by TRAST, in the NIR, with low autofluorescence and scattering conditions and on a broad range of samples and experimental conditions.

453 Rapid SARS-CoV-2 testing with duplexed recombinase polymerase amplification and a bacteriophage internal control

OBJECTIVES/GOALS: Current COVID-19 rapid molecular tests require cartridge-reader detection, expensive circuitry, and complex microfluidics making the most accurate tests unavailable to the masses. Here we present a rapid molecular diagnostic leveraging isothermal amplification and paper-based microfluidics for a low-cost ultra-sensitive COVID-19 assay. METHODS/STUDY POPULATION: We designed a reverse transcription recombinase polymerase amplification (RT-RPA) assay for the detection of SARS-CoV-2 and bacteriophage MS2 RNA. RT-RPA is a sequence specific, ultrasensitive, rapid isothermal DNA amplification technique that is well suited to home based testing due to its rapid assay time, robustness, ease of use, and readout options. RT-RPA reagents are added to a tube and incubated at 39°C in a fluorometer. Realtime fluorometer data gives results in under 15 minutes. This assay also provides visual detection via lateral flow readout with results in 23 minutes. RESULTS/ANTICIPATED RESULTS: We have developed a rapid multiplexed nucleic acid amplification assay with an internal process control for SARS-CoV-2 using single-pot RT-RPA. We screened 21 primer combinations to select primers that demonstrated excellent performance and target specificity against common respiratory viruses. We demonstrate the ability to multiplex SARS-CoV-2 and MS2 detection, utilizing MS2 as an internal process control for lysis, reverse transcription, amplification, and readout. We show duplexed detection using both fluorescence readout and visual readout using lateral flow strips. Duplexed fluorescence detection shows a limit of detection of 25 copies per reaction. Duplexed lateral flow readout shows a limit of detection of 50 copies per reaction DISCUSSION/SIGNIFICANCE: We developed a duplexed RT-RPA assay for SARS-CoV-2 with fluorescence or lateral flow readout. Our assay does not re-quire expensive reader, circuity, or fluid handling. The low material cost, temperature, and robustness make it ideal for a more accurate home-based COVID-19 diagnostic.

Liposome fusogenic enzyme-free circuit enables high-fidelity determination of single exosomal RNA

Accurate determination of single exosomal inclusions in situ presents a significant challenge due to their extremely low abundance as well sub-100 nm vesicle dimensions. Here, we created a Liposome Fusogenic Enzyme-free circuit (LIFE) approach for the high-fidelity identification of exosome-encapsulated cargoes without destroying the vesicle integrity. The probe-loaded cationic fusogenic liposome could capture and fuse with a single target exosome, enabling probes delivery and target biomolecule-initiated cascaded signal amplification in situ. Then the DNAzyme probe encountered conformal change upon exosomal microRNA activation, and generated a convex DNAzyme structure to cleave the RNA site of substrate probe. After that, the target microRNA could be released to introduce a cleavage cycle to yield amplified fluorescence readout. Therefore, trace cargoes in a single exosome could be accurately determined by elaborately controlling the ratio of introduced LIFE probe, paving the way toward the exploration of a universal sensing platform for the assessment of exosomal cargoes to facilitate early disease diagnosis and personalized treatment.

Facile Label-Free Three-Input Molecular Keypad Lock

Featured with molecule-level data encryption, molecular keypad locks show attractive merits in information security. Most of the previous multiple-input locks use fluorescence as output but are impeded by inefficient/labile prequenching or highly synthetic complexity/difficulty of the fluorophore-containing processor molecules. We herein propose a facile three-input molecular keypad lock, which is simple in synthesis and label free but capable of in situ generation of a fluorescent moiety (dityrosine) for background-free fluorescence readout. A nonfluorescent ("Locked") tyrosine derivate zYpc was easily synthesized as the processor. The correct "password" (i.e., UV → ALP → TYR, ABC) stepwise converted zYpc to a dityrosine-containing product, exhibiting a bright blue fluorescence output ("Open"). In contrast, wrongly permutated inputs failed to open this lock. This device shows potential to be extended as a more advanced keypad lock with better security.

Cas12a/Guide RNA-Based Platforms for Rapidly and Accurately Identifying Staphylococcus aureus and Methicillin-Resistant S. aureus.

In order to ensure the prevention and control of methicillin-resistant Staphylococcus aureus (MRSA) infection, rapid and accurate detection of pathogens and their resistance phenotypes is a must. Therefore, this study aimed to develop a fast and precise nucleic acid detection platform for identifying S. aureus and MRSA. We initially constructed a CRISPR-Cas12a detection system by designing single guide RNAs (sgRNAs) specifically targeting the thermonuclease (nuc) and mecA genes. To increase the sensitivity of the CRISPR-Cas12a system, we incorporated PCR, loop-mediated isothermal amplification (LAMP), and recombinase polymerase amplification (RPA). Subsequently, we compared the sensitivity and specificity of the three amplification methods paired with the CRISPR-Cas12a system. Finally, the clinical performance of the methods was tested by analyzing the fluorescence readout of 111 clinical isolates. In order to visualize the results, lateral-flow test strip technology, which enables point-of-care testing, was also utilized. After comparing the sensitivity and specificity of three different methods, we determined that the nuc-LAMP-Cas12a and mecA-LAMP-Cas12a methods were the optimal detection methods. The nuc-LAMP-Cas12a platform showed a limit of detection (LOD) of 10 aM (~6 copies μL-1), while the mecA-LAMP-Cas12a platform demonstrated a LOD of 1 aM (~1 copy μL-1). The LOD of both platforms reached 4 × 103 fg/μL of genomic DNA. Critical evaluation of their efficiencies on 111 clinical bacterial isolates showed that they were 100% specific and 100% sensitive with both the fluorescence readout and the lateral-flow readout. Total detection time for the present assay was approximately 80 min (based on fluorescence readout) or 85 min (based on strip readout). These results indicated that the nuc-LAMP-Cas12a and mecA-LAMP-Cas12a platforms are promising tools for the rapid and accurate identification of S. aureus and MRSA. IMPORTANCE The spread of methicillin-resistant Staphylococcus aureus (MRSA) poses a major threat to global health. Isothermal amplification combined with the trans-cleavage activity of Cas12a has been exploited to generate diagnostic platforms for pathogen detection. Here, we describe the design and clinical evaluation of two highly sensitive and specific platforms, nuc-LAMP-Cas12a and mecA-LAMP-Cas12a, for the detection of S. aureus and MRSA in 111 clinical bacterial isolates. With a limit of detection (LOD) of 4 × 103 fg/μL of genomic DNA and a turnaround time of 80 to 85 min, the present assay was 100% specific and 100% sensitive using either fluorescence or the lateral-flow readout. The present assay promises clinical application for rapid and accurate identification of S. aureus and MRSA in limited-resource settings or at the point of care. Beyond S. aureus and MRSA, similar CRISPR diagnostic platforms will find widespread use in the detection of various infectious diseases, malignancies, pharmacogenetics, food contamination, and gene mutations.

Rhodamine-Sensitized Two-Photon Activation of a Red Light-Absorbing BODIPY Photocage.

Two-photon (2P) activatable probes are of high value in biological and medical chemistry since near infrared (NIR) light can penetrate deeply even in blood-perfused tissue and due to the intrinsic three-dimensional activation properties. Designing two-photon chromophores is challenging. However, the two-photon absorption qualities of a photocage can be improved with an intramolecular sensitizer, which transfers the absorbed light onto the cage. We herein present the synthesis and photophysical characterization of a 2P-sensitive uncaging dyad based on rhodamine 101 as donor fluorophore and a redshifted BODIPY as acceptor photocage. Liberation of p-nitroaniline (PNA) upon one-photon photolysis was confirmed by HPLC analysis. The photoreaction was found to be accompanied by a considerable change of the fluorescence properties of the chromophores. The possibility of a fluorescent read-out enabled the detection of two-photon induced uncaging by confocal fluorescence microscopy.

Glow-in-the-Dark Infectious Disease Diagnostics Using CRISPR-Cas9-Based Split Luciferase Complementation.

Nucleic acid detection methods based on CRISPR and isothermal amplification techniques show great potential for point-of-care diagnostic applications. However, most current methods rely on fluorescent or lateral flow assay readout, requiring external excitation or postamplification reaction transfer. Here, we developed a bioluminescent nucleic acid sensor (LUNAS) platform in which target dsDNA is sequence-specifically detected by a pair of dCas9-based probes mediating split NanoLuc luciferase complementation. LUNAS is easily integrated with recombinase polymerase amplification (RPA), providing attomolar sensitivity in a rapid one-pot assay. A calibrator luciferase is included for a robust ratiometric readout, enabling real-time monitoring of the RPA reaction using a simple digital camera. We designed an RT-RPA-LUNAS assay that allows SARS-CoV-2 RNA detection without the need for cumbersome RNA isolation and demonstrated its diagnostic performance for COVID-19 patient nasopharyngeal swab samples. Detection of SARS-CoV-2 from samples with viral RNA loads of ∼200 cp/μL was achieved within ∼20 min, showing that RPA-LUNAS is attractive for point-of-care infectious disease testing.

Organelle-selective click labeling coupled with flow cytometry allows pooled CRISPR screening of genes involved in phosphatidylcholine metabolism

Cellular lipid synthesis and transport are governed by intricate protein networks. Although genetic screening should contribute to deciphering the regulatory networks of lipid metabolism, technical challenges remain-especially for high-throughput readouts of lipid phenotypes. Here, we coupled organelle-selective click labeling of phosphatidylcholine (PC) with flow cytometry-based CRISPR screening technologies to convert organellar PC phenotypes into a simple fluorescence readout for genome-wide screening. This technique, named O-ClickFC, was successfully applied in genome-scale CRISPR-knockout screens to identify previously reported genes associated with PC synthesis (PCYT1A, ACACA), vesicular membrane trafficking (SEC23B, RAB5C), and non-vesicular transport (PITPNB, STARD7). Moreover, we revealed previously uncharacterized roles of FLVCR1 as a choline uptake facilitator, CHEK1 as a post-translational regulator of the PC-synthetic pathway, and CDC50A as responsible for the translocation of PC to the outside of the plasma membrane bilayer. These findings demonstrate the versatility of O-ClickFC as an unprecedented platform for genetic dissection of cellular lipid metabolism.

CRISPR/Cas12a bio-assay integrated with metal-organic framework based enhanced fluorescent labels for ultrasensitive detection of circulating tumor DNA

Circulating tumor DNA (ctDNA) is an important biomarker for liquid biopsy. Unfortunately, the detection of ctDNA is still challenging unless these obstacles, such as unsatisfactory sensitivity, time-consuming processes, and high costs, are solved. Herein, the first paradigm of nanomaterial’s application in CRISPR/Cas12a system with enhanced fluorescence readout was reported in this work. We designed and prepared a class of metal-organic framework (MOF) based fluorescent labels with stimulus response to control the release of fluorescent molecules. Notably, the MOF-based fluorescent labels was able to optimize traditional CRISPR/Cas12a detection system with fluorescence readout, which improved the detection sensitivity over four orders of magnitude to meet the requirements of trace ctDNA detection. We integrated the MOF-based fluorescent labels and CRISPR/Cas12a system to achieve ultrasensitive and convenient detection of target ctDNA. Aiming at Kirsten rat sarcoma-2 virus point mutations, the proposed strategy exhibited ultrahigh sensitivity for detecting target ctDNA as low as 5.6 fM, which had been successfully employed to quantify target ctDNA in clinical serum samples. The quantitative results could distinguish lung cancer patients from healthy person with high specificity and sensitivity, which had a broad application prospect in clinical diagnosis.

Construction of a simple dual-mode ATP-sensing system for reliable fish freshness evaluation

Adenosine triphosphate (ATP), the main carrier of chemical energy, plays a key role in various biochemical reactions such as cellular metabolism. Currently, ATP levels are considered important indicators of microbial content in food safety, and food freshness can be determined by detecting ATP content. Some ATP sensing strategies have been applied to evaluate food freshness. However, cumbersome nanomaterial preparation, low sensitivity, and low reliability hamper their widespread application. Herein, a simple, high-performance, and reliable dual-mode sensing system based on hemin-G-quadruplex (G4) DNAzyme was established to detect ATP and assess fish freshness. Two nucleic acid probes, including subunits of the hemin-G4 DNAzyme in inactive structures and anti-ATP aptamer, self-assemble upon the input of ATP into the active hemin-G4 DNAzyme unit. The generated DNAzyme acts as a biocatalyst for colorimetric or fluorescent readout of the sensing process. The colorimetric and fluorescent dual-mode sensing system enables highly sensitive and reliable analysis of target ATP with detection limits of 71 nM and 73 nM, respectively. Moreover, the biosensor exhibited good selectivity for differentiating ATP from other interfering analytes. The proposed system was used to detect ATP in perch samples, and a linear correlation between ATP level and microbial content was confirmed. The established ATP-sensing system reliably evaluated fish freshness. Notably, in comparison with microbiological counts, the proposed DNAzyme-based dual-mode strategy for freshness evaluation is facile, highly efficient, and cost-effective, thus providing a promising method for food safety and quality monitoring.

Single-Spin Readout and Quantum Sensing Using Optomechanically Induced Transparency.

Solid-state spin defects are promising quantum sensors for a large variety of sensing targets. Some of these defects couple appreciably to strain in the host material. We propose to use this strain coupling for mechanically mediated dispersive single-shot spin readout by an optomechanically induced transparency measurement. Surprisingly, the estimated measurement times for negatively charged silicon-vacancy defects in diamond are an order of magnitude shorter than those for single-shot optical fluorescence readout. Our scheme can also be used for general parameter-estimation metrology and offers a higher sensitivity than conventional schemes using continuous position detection.

Malachite Green-Based Detection of SARS-CoV-2 by One-Step Reverse Transcription Loop-Mediated Isothermal Amplification

The pandemic of severe acute respiratory syndrome 2 (SARS-CoV-2) revealed the necessity of diagnosis of the infected people to prevent the prevalence infection cycle. Many commercial pathogen diagnosis methods are based on the detection of genomic materials. Isothermal amplification methods such as loop-mediated-isothermal amplification (LAMP) are the method of choice in these cases. Reverse transcription steps are efficiently coupled to LAMP for the detection of pathogens with genomic RNAs such as SARS-CoV-2. Many detection systems for LAMP include fluorescent readout systems. Although such systems result in desirable limits of detection, the need for special instrumentation is the main dispute of such systems to become real point of care assays. In contrast, colorimetric detection methods would reduce costs and improve the applicability of the system. In this study one-step reverse transcription-LAMP reaction was established that enables visual detection of the SARS-CoV-2 genome. Nasopharyngeal RNA samples were first validated by reverse transcription quantitative polymerase chain reaction and then subjected to RT-LAMP. To lower the cost associated with the readout system equipment, malachite green (MG) was used. The color change of MG to blue allowed visual detection of the virus. Firstly, experiments were set up as two-step RT-LAMP reaction to identify the best primer sets. In addition, MG concentration was optimized with the significant colorimetric signal for the positive samples. Next, a one-step colorimetric method was developed for the detection of SARS-CoV-2 based on MG color shift in 2 h.

Unpicking DNA translocation in nanopores with simultaneous single-molecule fluorescence and optical single channel recording.

Nanopore-based DNA sequencing offers high throughput and long reads by measuring blockades in current as a strand is translocated through the pore, providing a single-molecule readout with high time resolution. However, electrical measurements alone offer only information regarding the current block itself; to improve nanopore sensing, a molecular picture of the steps of analyte binding, capture and translocation is required. Optical single channel recording of calcium flux through the nanopore enables parallel single-molecule capture events to be distinguished. We combine optical single channel recording with a single molecule fluorescence readout of DNA unzipping to correlate the steps of individual translocations and image the location of the system components. By annealing short oligomers bearing fluorophore-quencher pairs to the translocating DNA strand, we map the process of nanopore capture, threading and unzipping.

Photo-inactivation disrupts rapid fluorescence readout of jGCaMP8 calcium indicators tethered to Orai1 channels.

We sought to optimize indicator response time in channel-tethered genetically encoded Ca2+ indicators (GECIs) using the jGCaMP8 series indicators released by the HHMI Genie Project. These indicators possess <10 ms rise times, substantially narrowing the temporal resolution gap between electrical and optical recording of Ca2+ currents. DNA encoding jGCaMP8f was inserted downstream of the Orai1 coding sequence to monitor Orai1 Ca2+ channel activity using TIRF microscopy combined with whole-cell recording. The use of non-inactivating channel currents provided a steady and electrically controlled source of Ca2+ to compare current and fluorescence readouts of Orai1 channel activity in transfected HEK 293A cells. Non-inactivating test pulses to −40 mV produced fluorescence traces that unexpectedly declined by ∼25% over 100 ms before reaching a stable plateau. Moreover, using an Orai1 Y80E mutant that lacks fast Ca2+-dependent inactivation, fluorescence traces declined by ∼50% at −100 mV. Testing of Orai1-jGCaMP8f using unroofed cells demonstrated that rapid and partial fluorescence inactivation is a property of the indicator itself, rather than channel function. Photo-inactivation spontaneously recovered over 5 minutes, and recovery was accelerated in the absence of Ca2+. Mutational analysis of residues near the tri-peptide fluorophore of jGCaMP8f pointed to a mechanism: Q69M/C70V greatly increased (∼90%) photo-inactivation reminiscent of fluorescent protein fluorophore cis-trans photo-switching. Indeed, 405 nm illumination of jGCaMP8f or 8m/8s/6f led to immediate photo-recovery, and simultaneous illumination with 405 and 488 nm light blocked photo-inactivation. Subsequent mutagenesis produced a variant that lacks photo-inactivation but largely preserves the desirable properties of jGCaMP8f. Our results point to caution in interpreting rapidly changing Ca2+ signals using jGCaMP8 indicators, suggest strategies to avoid photo-switching, and serve as a starting point to produce more photo-stable, and thus more accurate, GECI derivatives.

Non-volatile optical memory based on cooperative orientation switching: improvement of recording speed and contrast by utilizing out-of-plane orientation mode.

Herein, we report a novel strategy toward non-volatile optical memory with high-contrast, high-speed recording, and non-destructive readout capability based on the cooperative out-of-plane orientation of a fluorescent dye doped into azobenzene liquid crystalline polymer film. By employing the out-of-plane orientation switching upon irradiation with UV light and thermal heating, high-contrast turn-on fluorescence switching was successfully achieved and the optical recording was demonstrated with non-destructive fluorescence readout capability. Furthermore, the recording speed and the fluorescence on/off contrast in the present system were dramatically improved compared to the previous in-plane orientation mode.

Genetically Encoded RNA-Based Bioluminescence Resonance Energy Transfer (BRET) Sensors.

RNA-based nanostructures and molecular devices have become popular for developing biosensors and genetic regulators. These programmable RNA nanodevices can be genetically encoded and modularly engineered to detect various cellular targets and then induce output signals, most often a fluorescence readout. Although powerful, the high reliance of fluorescence on the external excitation light raises concerns about its high background, photobleaching, and phototoxicity. Bioluminescence signals can be an ideal complementary readout for these genetically encoded RNA nanodevices. However, RNA-based real-time bioluminescent reporters have been rarely developed. In this study, we reported the first type of genetically encoded RNA-based bioluminescence resonance energy transfer (BRET) sensors that can be used for real-time target detection in living cells. By coupling a luciferase bioluminescence donor with a fluorogenic RNA-based acceptor, our BRET system can be modularly designed to image and detect various cellular analytes. We expect that this novel RNA-based bioluminescent system can be potentially used broadly in bioanalysis and nanomedicine for engineering biosensors, characterizing cellular RNA-protein interactions, and high-throughput screening or in vivo imaging.