A chemogenetic approach for dopamine imaging with tunable sensitivity.

Genetically-encoded dopamine (DA) sensors enable high-resolution imaging of DA release, but their ability to detect a wide range of extracellular DA levels, especially tonic versus phasic DA release, is limited by their intrinsic affinity. Here we show that a human-selective dopamine receptor positive allosteric modulator (PAM) can be used to boost sensor affinity on-demand. The PAM enhances DA detection sensitivity across experimental preparations (in vitro, ex vivo and in vivo) via one-photon or two-photon imaging. In vivo photometry-based detection of optogenetically-evoked DA release revealed that DETQ administration produces a stable 31 minutes window of potentiation without effects on animal behavior. The use of the PAM revealed region-specific and metabolic state-dependent differences in tonic DA levels and enhanced single-trial detection of behavior-evoked phasic DA release in cortex and striatum. Our chemogenetic strategy can potently and flexibly tune DA imaging sensitivity and reveal multi-modal (tonic/phasic) DA signaling across preparations and imaging approaches.

Compact photometric detector integrated with separation microchip for potential portable liquid chromatography system.

In recent years, miniaturized analytical instruments have been developing to meet the needs of portable and rapid analysis. The key of miniaturized analytical equipment is the miniaturization and integration of functional modules. This paper aims to develop a miniaturized photometric detector and separation microfluidic chip for a liquid chromatography (LC) system. The detector uses a light-emitting diode to emit ultraviolet light, which is collimated by an internal double lens. A Z-shaped flow cell with a long optical path is designed and fabricated in the separation microfluidic chip with a three-layer structure, which provides a tubing-free connection between the separation and detection unit. Detector performance is evaluated using hemoglobin (Hb) samples, with an upper limit of detection linearity (95 %) of 0.345 AU and stray light level as low as 0.08 %. Additionally, the microchip channel can be filled with cation exchange resin and C18 particles. Finally, an ion LC system and a reversed-phase LC system were constructed based on the miniaturized photometric detector and two microchips with different packed columns, respectively, and were successfully used in the separation and detection of two metabolic markers (glycated hemoglobin or bilirubin). The results of this study are expected to facilitate the development of a portable LC system and their application in community health services and family health management of chronic diseases.

Accuracy of a new photometric jaw tracking system in the frontal plane at different recording distances: An in-vitro study.

OBJECTIVES: To evaluate the accuracy of a new photometric jaw tracking system (JTS) in recording linear vertical movements in the frontal plane at different distances. METHODS: A mandibular plaster cast of a patient was placed on a simulation machine capable of linear movements along two spatial axes. Cyclops JTS (Itaka) was adapted to the plaster cast, while the head frame was attached to the simulation machine. The latter performed five linear movements from 20 to 40 mm in the y-axis; each movement was repeated five times at five different recording distance (380 to 420 mm). The recorded movements were measured and compared with those obtained with a laser Doppler vibrometer (LDV) for accuracy analysis. Data were statistically processed (α = 0.05). RESULTS: No statistically significant differences were found between Cyclops and LDV measurements on the y- and z-axes (p = 0.5). Changes in linear vertical motion and distance positions did not affect the accuracy, which remained relatively constant with similar trends and values less than 1 % for each parameter variation. The best condition observed was linear vertical movement of 30 mm at 420 mm (0.010 ± 0.023 mm). CONCLUSIONS: Cyclops has proven to be an accurate JTS in recording linear vertical movements in the frontal plane at different recording distances. For optimal recordings, the scanner should be placed as close as possible to the markers; excessive vertical movements decreased the accuracy. However, this study has limitations and requires in-vivo confirmations. CLINICAL SIGNIFICANCE: The tested JTS proved accurate in recording linear vertical movements in the frontal plane. However, given the limitations of the study, further investigation under real conditions is needed to support prosthetic and gnathological rehabilitations.

Identifying behavioral links to neural dynamics of multifiber photometry recordings in a mouse social behavior network.

Objective.Distributed hypothalamic-midbrain neural circuits help orchestrate complex behavioral responses during social interactions. Given rapid advances in optical imaging, it is a fundamental question how population-averaged neural activity measured by multi-fiber photometry (MFP) for calcium fluorescence signals correlates with social behaviors is a fundamental question. This paper aims to investigate the correspondence between MFP data and social behaviors.Approach:We propose a state-space analysis framework to characterize mouse MFP data based on dynamic latent variable models, which include a continuous-state linear dynamical system and a discrete-state hidden semi-Markov model. We validate these models on extensive MFP recordings during aggressive and mating behaviors in male-male and male-female interactions, respectively.Main results:Our results show that these models are capable of capturing both temporal behavioral structure and associated neural states, and produce interpretable latent states. Our approach is also validated in computer simulations in the presence of known ground truth.Significance:Overall, these analysis approaches provide a state-space framework to examine neural dynamics underlying social behaviors and reveals mechanistic insights into the relevant networks.

Protocol for fiber photometry recording from deep brain regions in head-fixed mice.

To exclude the influence of motion on in vivo calcium imaging, animals usually need to be fixed. However, the whole-body restraint can cause stress in animals, affecting experimental results. In addition, some brain regions are prone to bleeding during surgery, which lowers the success rate of calcium imaging. Here, we present a protocol for calcium imaging using heparin-treated fiber in head-fixed mice. We describe steps for stereotaxic surgery, including virus injection and optic fiber implantation, fiber photometry, and data analysis. For complete details on the use and execution of this protocol, please refer to Du et al.1.

Denaturing mass photometry for rapid optimization of chemical protein-protein cross-linking reactions.

Chemical cross-linking reactions (XL) are an important strategy for studying protein-protein interactions (PPIs), including low abundant sub-complexes, in structural biology. However, choosing XL reagents and conditions is laborious and mostly limited to analysis of protein assemblies that can be resolved using SDS-PAGE. To overcome these limitations, we develop here a denaturing mass photometry (dMP) method for fast, reliable and user-friendly optimization and monitoring of chemical XL reactions. The dMP is a robust 2-step protocol that ensures 95% of irreversible denaturation within only 5 min. We show that dMP provides accurate mass identification across a broad mass range (30 kDa-5 MDa) along with direct label-free relative quantification of all coexisting XL species (sub-complexes and aggregates). We compare dMP with SDS-PAGE and observe that, unlike the benchmark, dMP is time-efficient (3 min/triplicate), requires significantly less material (20-100×) and affords single molecule sensitivity. To illustrate its utility for routine structural biology applications, we show that dMP affords screening of 20 XL conditions in 1 h, accurately identifying and quantifying all coexisting species. Taken together, we anticipate that dMP will have an impact on ability to structurally characterize more PPIs and macromolecular assemblies, expected final complexes but also sub-complexes that form en route.

Tripedal DNA Walker as a Signal Amplifier Combined with a Potential-Resolved Multicolor Electrochemiluminescence Strategy for Ultrasensitive Detection of Prostate Cancer Staging Indicators.

A multicolor electrochemiluminescence (ECL) biosensor based on a closed bipolar electrode (BPE) array was proposed for the rapid and intuitive analysis of three prostate cancer staging indicators. First, [Irpic-OMe], [Ir(ppy)2(acac)], and [Ru(bpy)3]2+ were applied as blue, green, and red ECL emitters, respectively, whose mixed ECL emission colors covered the whole visible region by varying the applied voltages. Afterward, we designed a simple Mg2+-dependent DNAzyme (MNAzyme)-driven tripedal DNA walker (TD walker) to release three output DNAs. Immediately after, three output DNAs were added to the cathodic reservoirs of the BPE for incubation. After that, we found that the emission colors from the anode of the BPE changed as a driving voltage of 8.0 V was applied, mainly due to changes in the interfacial potential and faradaic currents at the two poles of the BPE. Via optimization of the experimental parameters, cutoff values of such three indicators at different clinical stages could be identified instantly with the naked eye, and standard precision swatches with multiple indicators could be prepared. Finally, in order to precisely determine the prostate cancer stage, the multicolor ECL device was used for clinical analysis, and the resulting images were then compared with standard swatches, laying the way for accurate prostate cancer therapy.

[Research on Dry Chemical Detection TechnologyBased on Reflectance Photometry].

In response to the issues of insufficient stability and accuracy in dry chemical detection using reflectance photometry, caused by the divergence and multiple internal reflections of the reflected light signal from the sample and the multilayer dry film test strip, a dry chemical reflectance photometry detection system based on an integrating sphere is designed. Firstly, an integrating sphere device is incorporated to reduce signal divergence and loss, ensuring even detection of the sample's reflected light signal and improving detection stability. Secondly, Light Tools optical simulation analysis is performed, and an integrating sphere detection model is established. Thirdly, the Williams-Clapper equation is employed to correct the error in reflectance density caused by multiple internal reflections, enhancing detection accuracy. Experimental validation demonstrates that the developed integrating sphere-based dry chemical reflectance photometry detection system improves the stability and accuracy of the detection system.

Confined DNA tetrahedral molecular sieve for size-selective electrochemiluminescence sensing.

Size selectivity is crucial in highly accurate preparation of biosensors. Herein, we described an innovative electrochemiluminescence (ECL) sensing platform based on the confined DNA tetrahedral molecular sieve (DTMS) for size-selective recognition of nucleic acids and small biological molecule. Firstly, DNA template (T) was encapsulated into the inner cavity of DNA tetrahedral scaffold (DTS) and hybridized with quencher (Fc) labeled probe DNA to prepare DTMS, accordingly inducing Ru(bpy)32+ and Fc closely proximate, resulting the sensor in a "signal-off" state. Afterwards, target molecules entered the cavity of DTMS to realize the size-selective molecular recognition while prohibiting large molecules outside of the DTMS, resulting the sensor in a "signal-on" state due to the release of Fc. The rigid framework structure of DTS and the anchor of DNA probe inside the DTS effectively avoided the nuclease degradation of DNA probe, and nonspecific protein adsorption, making the sensor possess potential application prospect for size-selective molecular recognition in diagnostic analysis with high accuracy and specificity.

Protocol for in vivo dual-color fiber photometry in the mouse thalamus.

In vivo calcium imaging of neural activity is an indispensable approach for understanding the mechanisms and functions of neural system. Development of advanced imaging tools and various genetically encoded calcium indicators allows us to simultaneously record the activity of different neural populations. Here, we present a protocol for acquiring neural activity of two discrete neural populations in mice using dual-color fiber photometry. We describe steps for injecting viral constructs and implanting the fiber optic through stereotaxic surgery, calcium signal acquisition, and data analysis. We also describe the incorporation of electroencephalogram and electromyography recordings with dual-color fiber photometry analysis. For complete details on the use and execution of this protocol, please refer to Shin et al.1.

Monitoring norepinephrine release in vivo using next-generation GRABNE sensors.

Norepinephrine (NE) is an essential biogenic monoamine neurotransmitter. The first-generation NE sensor makes in vivo, real-time, cell-type-specific and region-specific NE detection possible, but its low NE sensitivity limits its utility. Here, we developed the second-generation GPCR-activation-based NE sensors (GRABNE2m and GRABNE2h) with a superior response and high sensitivity and selectivity to NE both in vitro and in vivo. Notably, these sensors can detect NE release triggered by either optogenetic or behavioral stimuli in freely moving mice, producing robust signals in the locus coeruleus and hypothalamus. With the development of a novel transgenic mouse line, we recorded both NE release and calcium dynamics with dual-color fiber photometry throughout the sleep-wake cycle; moreover, dual-color mesoscopic imaging revealed cell-type-specific spatiotemporal dynamics of NE and calcium during sensory processing and locomotion. Thus, these new GRABNE sensors are valuable tools for monitoring the precise spatiotemporal release of NE in vivo, providing new insights into the physiological and pathophysiological roles of NE.

Combined Experiments with in Vivo Fiber Photometry and Behavior Tests Can Facilitate the Measurement of Neuronal Activity in the Primary Somatosensory Cortex and Hyperalgesia in an Inflammatory Pain Mice Model.

The pain matrix, which includes several brain regions that respond to pain sensation, contribute to the development of chronic pain. Thus, it is essential to understand the mechanism of causing chronic pain in the pain matrix such as anterior cingulate (ACC), or primary somatosensory (S1) cortex. Recently, combined experiment with the behavior tests and in vivo calcium imaging using fiber photometry revealed the interaction between the neuronal function in deep brain regions of the pain matrix including ACC and the phenotype of chronic pain. However, it remains unclear whether this combined experiment can identify the interaction between neuronal activity in S1, which receive pain sensation, and pain behaviors such as hyperalgesia or allodynia. In this study, to examine whether the interaction between change of neuronal activity in S1 and hyperalgesia in hind paw before and after causing inflammatory pain was detected from same animal, the combined experiment of in vivo fiber photometry system and von Frey hairs test was applied. This combined experiment detected that amplitude of calcium responses in S1 neurons increased and the mechanical threshold of hind paw decreased from same animals which have an inflammatory pain. Moreover, we found that the values between amplitude of calcium responses and mechanical thresholds were shifted to negative correlation after causing inflammatory pain. Thus, the combined experiment with fiber photometry and the behavior tests has a possibility that can simultaneously consider the interaction between neuronal activity in pain matrix and pain induced behaviors and the effects of analgesics or pain treatments.

Self-Assembled Tetraphenylethene-Based Nanoaggregates with Tunable Electrochemiluminescence for the Ultrasensitive Detection of E. coli.

As an effective ECL emitter, tetraphenylethene (TPE)-based molecules have recently been reported with aggregation-induced electrochemiluminescence (AIECL) property, while it is still a big challenge to control its aggregation states and obtain uniform aggregates with intense ECL emission. In this study, we develop three TPE derivatives carrying a pyridinium group, an alkyl chain, and a quaternary ammonium group via the Menschutkin reaction. The resulting molecules exhibit significantly red-shifted FL and enhanced ECL emissions due to the tunable reduction of the energy gap between the highest occupied molecular orbitals (HOMOs) and the lowest unoccupied molecular orbitals (LUMOs). More importantly, the amphiphilicity of the as-developed molecules enables their spontaneous self-assembly into well-controlled spherical nanoaggregates, and the ECL intensity of nanoaggregates with 3 -CH2- (named as C3) is 17.0-fold higher compared to that of the original 4-(4-(1,2,2-triphenylvinyl)phenyl)pyridine (TPP) molecule. These cationic nanoaggregates demonstrate a high affinity toward bacteria, and an ECL sensor for the profiling of Escherichia coli (E. coli) was developed with a broad linear range and good selectivity in the presence of an E. coli-specific aptamer. This study provides an effective way to enhance the ECL emission of TPE molecules through their derivatization and a simple way to prepare well-controlled AIECL nanoaggregates for ECL application.

Potential-Resolved Ratiometric Aptasensor for Sensitive Acetamiprid Analysis Based on Coreactant-free Electrochemiluminescence Luminophores of Gd-MOF and "Light Switch" Molecule of [Ru(bpy)2dppz]2.

For conventional potential-resolved ratiometric electrochemiluminescence (ECL) systems, the introduction of multiplex coreactants is imperative. However, the undesirable interactions between different coreactants inevitably affect analytical accuracy and sensitivity. Herein, through the coordination of aggregation-induced emission ligands with gadolinium cations, the self-luminescent metal-organic framework (Gd-MOF) is prepared and serves as a novel coreactant-free anodic ECL emitter. By the intercalation of [Ru(bpy)2dppz]2+ with light switch effect into DNA duplex, one high-efficiency cathodic ECL probe is obtained using K2S2O8 as a coreactant. In the presence of acetamiprid, the strong affinity between the target and its aptamer induces the release of [Ru(bpy)2dppz]2+, resulting in a decreasing cathode signal and an increasing anode signal owing to the ECL resonance energy transfer from Gd-MOF to [Ru(bpy)2dppz]2+. In this way, an efficient dual-signal ECL aptasensor is constructed for the ratiometric analysis of acetamiprid, exhibiting a remarkably low detection limit of 0.033 pM. Strikingly, by using only one exogenous coreactant, the cross interference from multiple coreactants can be eliminated, thus improving the detection accuracy. The developed high-performance ECL sensing platform is successfully applied to monitor the residual level of acetamiprid in real samples, demonstrating its potential application in the field of food security.

Metal-organic framework (MOF)-based biosensors for miRNA detection.

MicroRNAs (miRNAs) play several crucial roles in the physiological and pathological processes of the human body. They are considered as important biomarkers for the diagnosis of various disorders. Thus, rapid, sensitive, selective, and affordable detection of miRNAs is of great importance. However, the small size, low abundance, and highly similar sequences of miRNAs impose major challenges to their accurate detection in biological samples. In recent years, metal-organic frameworks (MOFs) have been applied as promising sensing materials for the fabrication of different biosensors due to their distinctive characteristics, such as high porosity and surface area, tunable pores, outstanding adsorption affinities, and ease of functionalization. In this review, the applications of MOFs and MOF-derived materials in the fabrication of fluorescence, electrochemical, chemiluminescence, electrochemiluminescent, and photoelectrochemical biosensors for the detection of miRNAs and their detection principle and analytical performance are discussed. This paper attempts to provide readers with a comprehensive knowledge of the fabrication and sensing mechanisms of miRNA detection platforms.

Portable, intelligent MIECL sensing platform for ciprofloxacin detection using a fast convolutional neural networks-assisted Tb@Lu2O3 nanoemitter.

Environmental pollution caused by ciprofloxacin is a major problem of global public health. A machine learning-assisted portable smartphone-based visualized molecularly imprinted electrochemiluminescence (MIECL) sensor was developed for the highly selective and sensitive detection of ciprofloxacin (CFX) in food. To boost the efficiency of electrochemiluminescence (ECL), oxygen vacancies (OVs) enrichment was introduced into the flower-like Tb@Lu2O3 nanoemitter. With the specific recognition reaction between MIP as capture probes and CFX as detection target, the ECL signal significantly decreased. According to, CFX analysis was determined by traditional ECL analyzer detector in the concentration range from 5 × 10-4 to 5 × 102 µmol L-1 with the detection limit (LOD) of 0.095 nmol L-1 (S/N = 3). Analysis of luminescence images using fast electrochemiluminescence judgment network (FEJ-Net) models, achieving portable and intelligent quick analysis of CFX. The proposed MIECL sensor was used for CFX analysis in real meat samples and satisfactory results, as well as efficient selectivity and good stability.

Dual-microRNA-Controlled Electrochemiluminescence Biosensor for Breast Cancer Diagnosis and Supplemental Identification of Breast Cancer Metastasis.

Breast cancer remains the most frequently diagnosed cancer globally, and the metastasis of this malignancy is the primary cause of mortality in breast cancer patients. Hence, prompt diagnosis and timely detection of metastatic breast cancer are critical for effective therapeutic intervention. Both progression and metastasis of this malignancy are closely associated with aberrant expression of specific microRNAs (miRNAs) and enzymes. To facilitate breast cancer diagnosis and concomitant identification of metastatic breast cancer, we have engineered an innovative electrochemiluminescence (ECL)-based sensing platform integrated with enzyme-free DNA amplification circuits for dual functionality. Specifically, microRNA-21 (miR-21) is employed as a biomarker for breast cancer, and miR-21 induces the quenching of the ECL signal from luminophores via a strategically designed catalytic three-hairpin assembly (CTHA) circuit. Subsequently, miR-105 levels are measured via toehold-mediated strand displacement reactions (TSDR). Here, miR-105 restores the initially quenched ECL signal, enabling the assessment of the metastatic propensity. Our experimental data demonstrate that the devised ECL biosensor offers broad linear detection ranges and low detection limits for both miR-21 and miR-105. Importantly, our novel platform was also successfully validated by using cellular and serum samples. This biosensor not only discriminates breast cancer cell lines MCF-7 and MDA-MB-231 from nonbreast cancer cells like HepG2, TPC-1, and HeLa, but it also distinguishes between malignant MCF-7 and metastatic MDA-MB-231 cells. Consequently, our novel approach holds significant promise for clinical applications and precise cancer screening.

Comprehensive Impurity Profiling of mRNA: Evaluating Current Technologies and Advanced Analytical Techniques.

In vitro transcription (IVT) of mRNA is a versatile platform for a broad range of biotechnological applications. Its rapid, scalable, and cost-effective production makes it a compelling choice for the development of mRNA-based cancer therapies and vaccines against infectious diseases. The impurities generated during mRNA production can potentially impact the safety and efficacy of mRNA therapeutics, but their structural complexity has not been investigated in detail yet. This study pioneers a comprehensive profiling of IVT mRNA impurities, integrating current technologies with innovative analytical tools. We have developed highly reproducible, efficient, and stability-indicating ion-pair reversed-phase liquid chromatography and capillary gel electrophoresis methods to determine the purity of mRNA from different suppliers. Furthermore, we introduced the applicability of microcapillary electrophoresis for high-throughput (<1.5 min analysis time per sample) mRNA impurity profiling. Our findings revealed that impurities are mainly attributed to mRNA variants with different poly(A) tail lengths due to aborted additions or partial hydrolysis and the presence of double-stranded mRNA (dsRNA) byproducts, particularly the dsRNA 3'-loop back form. We also implemented mass photometry and native mass spectrometry for the characterization of mRNA and its related product impurities. Mass photometry enabled the determination of the number of nucleotides of different mRNAs with high accuracy as well as the detection of their size variants [i.e., aggregates and partial and/or total absence of the poly(A) tail], thus providing valuable information on mRNA identity and integrity. In addition, native mass spectrometry provided insights into mRNA intact mass, heterogeneity, and important sequence features such as poly(A) tail length and distribution. This study highlights the existing bottlenecks and opportunities for improvement in the analytical characterization of IVT mRNA, thus contributing to the refinement and streamlining of mRNA production, paving the way for continued advancements in biotechnological applications.

Rigidifying AIEgens in covalent organic framework nanosheets for electrochemiluminescence enhancement: TABE-PZ-CON as a novel emitter for microRNA-21 detection.

Enhancing electrochemiluminescence (ECL) properties of luminophores is a hot direction in the current ECL field. Herein, we found that covalent rigidification of the aggregation-induced emission luminogens (AIEgens) TABE (TABE = tetra-(4-aldehyde-(1,1-biphenyl))ethylene) into covalent organic framework nanosheets (TABE-PZ-CON, PZ = piperazine) could result in stronger ECL emission than those of TABE aggregates and TABE monomers. We termed the interesting phenomenon "covalent rigidification-triggered electrochemiluminescence (CRT-ECL) enhancement". The superior ECL performance of TABE-PZ-CON not only because massive TABE luminogens were covalently assembled into the rigid TABE-PZ-CON network, which limited the intramolecular motions of TABE and hampered the radiationless transition, but also because the ultrathin porous TABE-PZ-CON significantly reduced the transportation distance of ions, electrons, and coreactants, which enabled the electrochemical excitation of more TABE luminogens and thus enhanced the ECL efficiency. Bearing in mind the exceptional ECL performance of TABE-PZ-CON, it was utilized as a high-efficient ECL indicator in combination with the DNA walker and duplex-specific nuclease-assisted target recycling amplification strategies to design an "off-on" ECL biosensor for the ultrasensitive assay of microRNA-21, exhibiting a favorable response range (100 aM-1 nM) with an ultralow detection limit of 17.9 aM. Overall, this work offers a valid way to inhibit the intramolecular motions of AIEgens for ECL enhancement, which gives a new vision for building high-performance AIEgen-based ECL materials, thus offering more chances for assembling hypersensitive ECL biosensors.

Analysis of Protein Complex Formation at Micromolar Concentrations by Coupling Microfluidics with Mass Photometry.

Mass photometry is a versatile mass measurement technology that enables the study of biomolecular interactions and complex formation in solution without labels. Mass photometry is generally suited to analyzing samples in the 100 pM-100 nM concentration range. However, in many biological systems, it is necessary to measure more concentrated samples to study low-affinity or transient interactions. Here, we demonstrate a method that effectively expands the range of sample concentrations that can be analyzed by mass photometry from nanomolar to tens of micromolar. In this protocol, mass photometry is combined with a novel microfluidics system to investigate the formation of protein complexes in solution in the micromolar concentration range. With the microfluidics system, users can maintain a sample at a desired higher concentration followed by dilution to the nanomolar range - several milliseconds prior to the mass photometry measurement. Due to the speed of the dilution, data is obtained before the equilibrium of the sample has shifted (i.e., dissociation of the complex). The technique is applied to measure interactions between an immunoglobulin G (IgG) antibody and the neonatal Fc receptor, showing the formation of high-order complexes that were not quantifiable with static mass photometry measurements. In conclusion, the combination of mass photometry and microfluidics makes it possible to characterize samples in the micromolar concentration range and is proficient in measuring biomolecular interactions with weaker affinities. These capabilities can be applied in a range of contexts - including the development and design of biotherapeutics - enabling thorough characterization of diverse protein-protein interactions.